Compositions and methods for WT1 specific immunotherapy

ABSTRACT

Compositions and methods for the therapy of malignant diseases, such as leukemia and cancer, are disclosed. The compositions comprise one or more of a WT1 polynucleotide, a WT1 polypeptide, an antigen-presenting cell presenting a WT1 polypeptide, an antibody that specifically binds to a WT1 polypeptide; or a T cell that specifically reacts with a WT1 polypeptide. Such compositions may be used, for example, for the prevention and treatment of metastatic diseases.

This invention was made in part with government support under NIH SBIR Phase I grant number IR43 CA81752-01A1. The Government may have certain rights in this invention.

TECHNICAL FIELD

The present invention relates generally to the immunotherapy of malignant diseases such as leukemia and cancers. The invention is more specifically related to compositions for generating or enhancing an immune response to WT1, and to the use of such compositions for preventing and/or treating malignant diseases.

BACKGROUND OF THE INVENTION

Cancer and leukemia are significant health problems in the United States and throughout the world. Although advances have been made in detection and treatment of such diseases, no vaccine or other universally successful method for prevention or treatment of cancer and leukemia is currently available. Management of the diseases currently relies on a combination of early diagnosis and aggressive treatment, which may include one or more of a variety of treatments such as surgery, radiotherapy, chemotherapy and hormone therapy. The course of treatment for a particular cancer is often selected based on a variety of prognostic parameters, including an analysis of specific tumor markers. However, the use of established markers often leads to a result that is difficult to interpret, and the high mortality continues to be observed in many cancer patients.

Immunotherapies have the potential to substantially improve cancer and leukemia treatment and survival. Recent data demonstrate that leukemia can be cured by immunotherapy in the context of bone marrow transplantation (e.g., donor lymphocyte infusions). Such therapies may involve the generation or enhancement of an immune response to a tumor-associated antigen (TAA). However, to date relatively few TAAs are known and the generation of an immune response against such antigens has, with rare exception, not been shown to be therapeutically beneficial.

Accordingly, there is a need in the art for improved methods for leukemia and cancer prevention and therapy. The present invention fulfills these needs and further provides other related advantages.

SUMMARY OF THE INVENTION

Briefly stated, this invention provides compositions and methods for the diagnosis and therapy of diseases such as leukemia and cancer. In one aspect, the present invention provides polypeptides comprising an immunogenic portion of a native WT1, or a variant thereof that differs in one or more substitutions, deletions, additions and/or insertions such that the ability of the variant to react with antigen-specific antisera and/or T-cell lines or clones is not substantially diminished. Within certain embodiments, the polypeptide comprises no more than 16 consecutive amino acid residues of a native WT1 polypeptide. Within other embodiments, the polypeptide comprises an immunogenic portion of amino acid residues 1–174 of a native WT1 polypeptide or a variant thereof, wherein the polypeptide comprises no more than 16 consecutive amino acid residues present within amino acids 175 to 449 of the native WT1 polypeptide. The immunogenic portion preferably binds to an MHC class I and/or class II molecule. Within certain embodiments, the polypeptide comprises a sequence selected from the group consisting of (a) sequences recited in any one or more of Tables II–XLVI, (b) variants of the foregoing sequences that differ in one or more substitutions, deletions, additions and/or insertions such that the ability of the variant to react with antigen-specific antisera and/or T-cell lines or clones is not substantially diminished and (c) mimetics of the polypeptides recited above, such that the ability of the mimetic to react with antigen-specific antisera and/or T cell lines or clones is not substantially diminished.

Within other embodiments, the polypeptide comprises a sequence selected from the group consisting of (a) ALLPAVPSL (SEQ ID NO:34), GATLKGVAA (SEQ ID NO:88), CMTWNQMNL (SEQ ID NOs: 49 and 258), SCLESQPTI (SEQ ID NOs: 199 and 296), SCLESQPAI (SEQ ID NO:198), NLYQMTSQL (SEQ ID NOs: 147 and 284), ALLPAVSSL (SEQ ID NOs: 35 and 255), RMFPNAPYL (SEQ ID NOs: 185 and 293), (b) variants of the foregoing sequences that differ in one or more substitutions, deletions, additions and/or insertions such that the ability of the variant to react with antigen-specific antisera and/or T-cell lines or clones is not substantially diminished and (c) mimetics of the polypeptides recited above, such that the ability of the mimetic to react with antigen-specific antisera and/or T cell lines or clones is not substantially diminished. Mimetics may comprises amino acids in combination with one or more amino acid mimetics or may be entirely nonpeptide mimetics.

Within further aspects, the present invention provides polypeptides comprising a variant of an immunogenic portion of a WT1 protein, wherein the variant differs from the immunogenic portion due to substitutions at between 1 and 3 amino acid positions within the immunogenic portion such that the ability of the variant to react with antigen-specific antisera and/or T-cell lines or clones is enhanced relative to a native WT1 protein.

The present invention further provides WT1 polynucleotides that encode a WT1 polypeptide as described above.

Within other aspects, the present invention provides pharmaceutical compositions and vaccines. Pharmaceutical compositions may comprise a polypeptide or mimetic as described above and/or one or more of (i) a WT1 polynucleotide; (ii) an antibody or antigen-binding fragment thereof that specifically binds to a WT1 polypeptide; (iii) a T cell that specifically reacts with a WT1 polypeptide or (iv) an antigen-presenting cell that expresses a WT1 polypeptide, in combination with a pharmaceutically acceptable carrier or excipient. Vaccines comprise a polypeptide as described above and/or one or more of (i) a WT1 polynucleotide, (ii) an antigen-presenting cell that expresses a WT1 polypeptide or (iii) an anti-idiotypic antibody, and a non-specific immune response enhancer. Within certain embodiments, less than 23 consecutive amino acid residues, preferably less than 17 amino acid residues, of a native WT1 polypeptide are present within a WT1 polypeptide employed within such pharmaceutical compositions and vaccines. The immune response enhancer may be an adjuvant. Preferably, an immune response enhancer enhances a T cell response.

The present invention further provides methods for enhancing or inducing an immune response in a patient, comprising administering to a patient a pharmaceutical composition or vaccine as described above. In certain embodiments, the patient is a human.

The present invention further provides methods for inhibiting the development of a malignant disease in a patient, comprising administering to a patient a pharmaceutical composition or vaccine as described above. Malignant diseases include, but are not limited to leukemias (e.g., acute myeloid, acute lymphocytic and chronic myeloid) and cancers (e.g., breast, lung, thyroid or gastrointestinal cancer or a melanoma). The patient may, but need not, be afflicted with the malignant disease, and the administration of the pharmaceutical composition or vaccine may inhibit the onset of such a disease, or may inhibit progression and/or metastasis of an existing disease.

The present invention further provides, within other aspects, methods for removing cells expressing WT1 from bone marrow and/or peripheral blood or fractions thereof, comprising contacting bone marrow, peripheral blood or a fraction of bone marrow or peripheral blood with T cells that specifically react with a WT1 polypeptide, wherein the step of contacting is performed under conditions and for a time sufficient to permit the removal of WT1 positive cells to less than 10%, preferably less than 5% and more preferably less than 1%, of the number of myeloid or lymphatic cells in the bone marrow, peripheral blood or fraction. Bone marrow, peripheral blood and fractions may be obtained from a patient afflicted with a disease associated with WT1 expression, or may be obtained from a human or non-human mammal not afflicted with such a disease.

Within related aspects, the present invention provides methods for inhibiting the development of a malignant disease in a patient, comprising administering to a patient bone marrow, peripheral blood or a fraction of bone marrow or peripheral blood prepared as described above. Such bone marrow, peripheral blood or fractions may be autologous, or may be derived from a related or unrelated human or non-human animal (e.g., syngeneic or allogeneic).

In other aspects, the present invention provides methods for stimulating (or priming) and/or expanding T cells, comprising contacting T cells with a WT1 polypeptide under conditions and for a time sufficient to permit the stimulation and/or expansion of T cells. Such T cells may be autologous, allogeneic, syngeneic or unrelated WT1-specific T cells, and may be stimulated in vitro or in vivo. Expanded T cells may, within certain embodiments, be present within bone marrow, peripheral blood or a fraction of bone marrow or peripheral blood, and may (but need not) be clonal. Within certain embodiments, T cells may be present in a mammal during stimulation and/or expansion. WT1-specific T cells may be used, for example, within donor lymphocyte infusions.

Within related aspects, methods are provided for inhibiting the development of a malignant disease in a patient, comprising administering to a patient T cells prepared as described above. Such T cells may, within certain embodiments, be autologous, syngeneic or allogeneic.

The present invention further provides, within other aspects, methods for monitoring the effectiveness of an immunization or therapy for a malignant disease associated with WT1 expression in a patient. Such methods are based on monitoring antibody, CD4+ T cell and/or CD8+ T cell responses in the patient. Within certain such aspects, a method may comprise the steps of: (a) incubating a first biological sample with one or more of: (i) a WT1 polypeptide; (ii) a polynucleotide encoding a WT1 polypeptide; or (iii) an antigen presenting cell that expresses a WT1 polypeptide, wherein the first biological sample is obtained from a patient prior to a therapy or immunization, and wherein the incubation is performed under conditions and for a time sufficient to allow immunocomplexes to form; (b) detecting immunocomplexes formed between the WT1 polypeptide and antibodies in the biological sample that specifically bind to the WT1 polypeptide; (c) repeating steps (a) and (b) using a second biological sample obtained from the same patient following therapy or immunization; and (d) comparing the number of immunocomplexes detected in the first and second biological samples, and therefrom monitoring the effectiveness of the therapy or immunization in the patient.

Within certain embodiments of the above methods, the step of detecting comprises (a) incubating the immunocomplexes with a detection reagent that is capable of binding to the immunocomplexes, wherein the detection reagent comprises a reporter group, (b) removing unbound detection reagent, and (c) detecting the presence or absence of the reporter group. The detection reagent may comprise, for example, a second antibody, or antigen-binding fragment thereof, capable of binding to the antibodies that specifically bind to the WT1 polypeptide or a molecule such as Protein A. Within other embodiments, a reporter group is bound to the WT1 polypeptide, and the step of detecting comprises removing unbound WT1 polypeptide and subsequently detecting the presence or absence of the reporter group.

Within further aspects, methods for monitoring the effectiveness of an immunization or therapy for a malignant disease associated with WT1 expression in a patient may comprise the steps of: (a) incubating a first biological sample with one or more of: (i) a WT1 polypeptide; (ii) a polynucleotide encoding a WT1 polypeptide; or (iii) an antigen presenting cell that expresses a WT1 polypeptide, wherein the biological sample comprises CD4+ and/or CD8+ T cells and is obtained from a patient prior to a therapy or immunization, and wherein the incubation is performed under conditions and for a time sufficient to allow specific activation, proliferation and/or lysis of T cells; (b) detecting an amount of activation, proliferation and/or lysis of the T cells; (c) repeating steps (a) and (b) using a second biological sample comprising CD4+ and/or CD8+ T cells, wherein the second biological sample is obtained from the same patient following therapy or immunization; and (d) comparing the amount of activation, proliferation and/or lysis of T cells in the first and second biological samples, and therefrom monitoring the effectiveness of the therapy or immunization in the patient.

The present invention further provides methods for inhibiting the development of a malignant disease associated with WT1 expression in a patient, comprising the steps of: (a) incubating CD4⁺ and/or CD8+ T cells isolated from a patient with one or more of: (i) a WT1 polypeptide; (ii) a polynucleotide encoding a WT1 polypeptide; or (iii) an antigen presenting cell that expresses a WT1 polypeptide, such that the T cells proliferate; and (b) administering to the patient an effective amount of the proliferated T cells, and therefrom inhibiting the development of a malignant disease in the patient. Within certain embodiments, the step of incubating the T cells may be repeated one or more times.

Within other aspects, the present invention provides methods for inhibiting the development of a malignant disease associated with WT1 expression in a patient, comprising the steps of: (a) incubating CD4⁺ and/or CD8+ T cells isolated from a patient with one or more of: (i) a WT1 polypeptide; (ii) a polynucleotide encoding a WT1 polypeptide; or (iii) an antigen presenting cell that expresses a WT1 polypeptide, such that the T cells proliferate; (b) cloning one or more cells that proliferated; and (c) administering to the patient an effective amount of the cloned T cells.

Within other aspects, methods are provided for determining the presence or absence of a malignant disease associated with WT1 expression in a patient, comprising the steps of: (a) incubating CD4⁺ and/or CD8+ T cells isolated from a patient with one or more of: (i) a WT1 polypeptide; (ii) a polynucleotide encoding a WT1 polypeptide; or (iii) an antigen presenting cell that expresses a WT1 polypeptide; and (b) detecting the presence or absence of specific activation of the T cells, therefrom determining the presence or absence of a malignant disease associated with WT1 expression. Within certain embodiments, the step of detecting comprises detecting the presence or absence of proliferation of the T cells.

Within further aspects, the present invention provides methods for determining the presence or absence of a malignant disease associated with WT1 expression in a patient, comprising the steps of: (a) incubating a biological sample obtained from a patient with one or more of: (i) a WT1 polypeptide; (ii) a polynucleotide encoding a WT1 polypeptide; or (iii) an antigen presenting cell that expresses a WT1 polypeptide, wherein the incubation is performed under conditions and for a time sufficient to allow immunocomplexes to form; and (b) detecting immunocomplexes formed between the WT1 polypeptide and antibodies in the biological sample that specifically bind to the WT1 polypeptide; and therefrom determining the presence or absence of a malignant disease associated with WT1 expression.

These and other aspects of the present invention will become apparent upon reference to the following detailed description and attached drawings. All references disclosed herein are hereby incorporated by reference in their entirety as if each was incorporated individually.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a comparison of the mouse (MO) and human (HU) WT1 protein sequences (SEQ ID NOS: 320 and 319 respectively).

FIG. 2 is a Western blot illustrating the detection of WT1 specific antibodies in patients with hematological malignancy (AML). Lane 1 shows molecular weight markers; lane 2 shows a positive control (WT1 positive human leukemia cell line immunoprecipitated with a WT1 specific antibody); lane 3 shows a negative control (WT1 positive cell line immunoprecipitated with mouse sera); and lane 4 shows a WT1 positive cell line immunoprecipitated with sera of a patient with AML. For lanes 2–4, the immunoprecipitate was separated by gel electrophoresis and probed with a WT1 specific antibody.

FIG. 3 is a Western blot illustrating the detection of a WT1 specific antibody response in B6 mice immunized with TRAMP-C, a WT1 positive tumor cell line. Lanes 1, 3 and 5 show molecular weight markers, and lanes 2, 4 and 6 show a WT1 specific positive control (N180, Santa Cruz Biotechnology, polypeptide spanning 180 amino acids of the N-terminal region of the WT1 protein, migrating on the Western blot at 52 kD). The primary antibody used was WT180 in lane 2, sera of non-immunized B6 mice in lane 4 and sera of the immunized B6 mice in lane 6.

FIG. 4 is a Western blot illustrating the detection of WT1 specific antibodies in mice immunized with representative WT1 peptides. Lanes 1, 3 and 5 show molecular weight markers and lanes 2, 4 and 6 show a WT1 specific positive control (N180, Santa Cruz Biotechnology, polypeptide spanning 180 amino acids of the N-terminal region of the WT1 protein, migrating on the Western blot at 52 kD). The primary antibody used was WT180 in lane 2, sera of non-immunized B6 mice in lane 4 and sera of the immunized B6 mice in lane 6.

FIGS. 5A to 5C are graphs illustrating the stimulation of proliferative T cell responses in mice immunized with representative WT1 peptides. Thymidine incorporation assays were performed using one T cell line and two different clones, as indicated, and results were expressed as cpm. Controls indicated on the x axis were no antigen (No Ag) and B6/media; antigens used were p6–22 human (p1), p117–139 (p2) or p244–262 human (p3).

FIGS. 6A and 6B are histograms illustrating the stimulation of proliferative T cell responses in mice immunized with representative WT1 peptides. Three weeks after the third immunization, spleen cells of mice that had been inoculated with Vaccine A or Vaccine B were cultured with medium alone (medium) or spleen cells and medium (B6/no antigen), B6 spleen cells pulsed with the peptides p6–22 (p6), p117–139 (p117), p244–262 (p244) (Vaccine A; FIG. 6A) or p287–301 (p287), p299–313 (p299), p421–435 (p421) (Vaccine B; FIG. 6B) and spleen cells pulsed with an irrelevant control peptide (irrelevant peptide) at 25 ug/ml and were assayed after 96 hr for proliferation by (³H) thymidine incorporation. Bars represent the stimulation index (SI), which is calculated as the mean of the experimental wells divided by the mean of the control (B6 spleen cells with no antigen).

FIGS. 7A–7D are histograms illustrating the generation of proliferative T-cell lines and clones specific for p 117–139 and p6–22. Following in vivo immunization, the initial three in vitro stimulations (IVS) were carried out using all three peptides of Vaccine A or B, respectively. Subsequent IVS were carried out as single peptide stimulations using only the two relevant peptides p117–139 and p6-22. Clones were derived from both the p6–22 and p117–139 specific T cell lines, as indicated. T cells were cultured with medium alone (medium) or spleen cells and medium (B6/no antigen), B6 spleen cells pulsed with the peptides p6-22 (p6), pll7–139 (p117) or an irrelevant control peptide (irrelevant peptide) at 25 ug/ml and were assayed after 96 hr for proliferation by (³H) thymidine incorporation. Bars represent the stimulation index (SI), which is calculated as the mean of the experimental wells divided by the mean of the control (B6 spleen cells with no antigen).

FIGS. 8A and 8B present the results of TSITES Analysis of human WT1 (SEQ ID NO:319) for peptides that have the potential to elicit Th responses. Regions indicated by “A” are AMPHI midpoints of blocks, “R” indicates residues matching the Rothbard/'Taylor motif, “D” indicates residues matching the IAd motif, and ‘d’ indicates residues matching the IEd motif.

FIGS. 9A and 9B are graphs illustrating the elicitation of WT1 peptide-specific CTL in mice immunized with WT1 peptides. FIG. 9A illustrates the lysis of target cells by allogeneic cell lines and FIG. 9B shows the lysis of peptide coated cell lines. In each case, the % lysis (as determined by standard chromium release assays) is shown at three indicated effector:target ratios. Results are provided for lymphoma cells (LSTRA and E10), as well as E10+p235–243 (E10+P235). E10 cells are also referred to herein as EL-4 cells.

FIGS. 10A–10D are graphs illustrating the elicitation of WT1 specific CTL, which kill WT1 positive tumor cell lines but do not kill WT1 negative cell lines, following vaccination of B6 mice with WT1 peptide P117. FIG. 10A illustrates that T-cells of non-immunized B6 mice do not kill WT1 positive tumor cell lines. FIG. 10B illustrates the lysis of the target cells by allogeneic cell lines. FIGS. 10C and 10D demonstrate the lysis of WT1 positive tumor cell lines, as compared to WT1 negative cell lines in two different experiments. In addition, FIGS. 10C and 10D show the lysis of peptide-coated cell lines (WT1 negative cell line E10 coated with the relevant WT1 peptide P117) In each case, the % lysis (as determined by standard chromium release assays) is shown at three indicated effector:target ratios. Results are provided for lymphoma cells (E10), prostate cancer cells (TRAMP-C), a transformed fibroblast cell line (BLK-SV40), as well as E10+p117.

FIGS. 11A and 11B are histograms illustrating the ability of representative peptide P117–139 specific CTL to lyse WT1 positive tumor cells. Three weeks after the third immunization, spleen cells of mice that had been inoculated with the peptides p235–243 or p117–139 were stimulated in vitro with the relevant peptide and tested for ability to lyse targets incubated with WT1 peptides as well as WT1 positive and negative tumor cells. The bars represent the mean % specific lysis in chromium release assays performed in triplicate with an E:T ratio of 25:1. FIG. 11A shows the cytotoxic activity of the p235–243 specific T cell line against the WT 1 negative cell line EL-4 (EL-4, WT1 negative); EL-4 pulsed with the relevant (used for immunization as well as for restimulation) peptide p235–243 (EL-4+p235); EL-4 pulsed with the irrelevant peptides p117–139 (EL-4+p117) p126–134 (EL-4+p126) or p130–138 (EL-4+p130) and the WT1 positive tumor cells BLK-SV40 (BLK-SV40, WT1 positive) and TRAMP-C (TRAMP-C, WT1 positive), as indicated. FIG. 11B shows cytotoxic activity of the p117–139 specific T cell line against EL-4; EL-4 pulsed with the relevant peptide P117–139 (EL-4+p117) and EL-4 pulsed with the irrelevant peptides p123–131 (EL-4+p123), or p128–136 (EL-4+p128); BLK-SV40 and TRAMP-C, as indicated.

FIGS. 12A and 12B are histograms illustrating the specificity of lysis of WT1 positive tumor cells, as demonstrated by cold target inhibition. The bars represent the mean % specific lysis in chromium release assays performed in triplicate with an E:T ratio of 25:1. FIG. 12A shows the cytotoxic activity of the p117–139 specific T cell line against the WT1 negative cell line EL-4 (EL-4, WT1 negative); the WT1 positive tumor cell line TRAMP-C (TRAMP-C, WT1 positive); TRAMP-C cells incubated with a ten-fold excess (compared to the hot target) of EL-4 cells pulsed with the relevant peptide p117–139 (TRAMP-C+p117 cold target) without ⁵¹Cr labeling and TRAMP-C cells incubated with EL-4 pulsed with an irrelevant peptide without ⁵¹Cr labeling (TRAMP-C+irrelevant cold target), as indicated. FIG. 12B shows the cytotoxic activity of the p117–139 specific T cell line against the WT1 negative cell line EL-4 (EL-4, WT1 negative); the WT1 positive tumor cell line BLK-SV40 (BLK-SV40, WT1 positive); BLK-SV40 cells incubated with the relevant cold target (BLK-SV40+p117 cold target) and BLK-SV40 cells incubated with the irrelevant cold target (BLK-SV40+irrelevant cold target), as indicated.

FIGS. 13A–13C are histograms depicting an evaluation of the 9mer CTL epitope within p117–139. The p117–139 tumor specific CTL line was tested against peptides within aa117–139 containing or lacking an appropriate H-2^(b) class I binding motif and following restimulation with p126–134 or p130–138. The bars represent the mean % specific lysis in chromium release assays performed in triplicate with an E:T ratio of 25:1. FIG. 13A shows the cytotoxic activity of the p117–139 specific T cell line against the WT1 negative cell line EL-4 (EL-4, WT1 negative) and EL-4 cells pulsed with the peptides p117–139 (EL-4+p117), p119–127 (EL-4+p119), p120–128 (EL-4+p120(EL-4+p123), p126–134 (EL-4+p126), p128–136 (EL-4+p128), and p130–138 (EL-4+p130). FIG. 13B shows the cytotoxic activity of the CTL line after restimulation with p126–134 against the WT1 negative cell line EL-4, EL-4 cells pulsed with p117–139 (EL-4+p117), p126–134 (EL-4+p126) and the WT1 positive tumor cell line TRAMP-C. FIG. 13C shows the cytotoxic activity of the CTL line after restimulation with p130–138 against EL-4, EL-4 cells pulsed with p117–139 (EL-4+p117), p130–138 (EL-4+p130) and the WT1 positive tumor cell line TRAMP-C.

FIG. 14 depicts serum antibody reactivity to WT1 in 63 patients with AML. Reactivity of serum antibody to WT1/N-terminus protein was evaluated by ELISA in patients with AML. The first and second lanes represent the positive and negative controls, respectively. The first and second lanes represent the ositive and negative controls, respectively. Commercially obtained WT1 specific antibody WT180 was used for the positive control. The next 63 lanes represent results using sera from each individual patient. The OD values depicted were from ELISA using a 1:500 serum dilution. The figure includes cumulative data from 3 separate experiments.

FIG. 15 depicts serum antibody reactivity to WT1 proteins and control proteins in 2 patients with AML. Reactivity of serum antibody to WT1/full-length, WT1N-terminus, TRX and Ra12 proteins was evaluated by ELISA in 2 patients with AML. The OD values depicted were from ELISA using a 1:500 serum dilution. AML-1 and AML-2 denote serum from 2 of the individual patients in FIG. 1 with demonstrated antibody reactivity to WT1/full-length. The WT1 full-length protein was expressed as a fusion protein with Ra12. The WT1/N-terminus protein was expressed as a fusion protein with TRX. The control Ra12 and TRX proteins were purified in a similar manner. The results confirm that the serum antibody reactivity against the WT1 fusion proteins is directed against the WT1 portions of the protein.

FIG. 16 depicts serum antibody reactivity to WT1 in 81 patients with CML. Reactivity of serum antibody to WT1/full-length protein was evaluated by ELISA in patients with AML. The first and second lanes represent the positive and negative controls, respectively. Commercially obtained WT1 specific antibody WT180 was used for the positive control. The next 81 lanes represent results using sera from each individual patient. The OD values depicted were from ELISA using a 1:500 serum dilution. The figure includes cumulative data from 3 separate experiments.

FIG. 17 depicts serum antibody reactivity to WT1 proteins and control proteins in 2 patients with CML. Reactivity of serum antibody to WT1/full-length, WT1/N-terminus, TRX and Ra12 proteins was evaluated by ELISA in 2 patients with CML. The OD values depicted were from ELISA using a 1:500 serum dilution. CML-1 and CML-2 denote serum from 2 of the individual patients in FIG. 3 with demonstrated antibody reactivity to WT1/full-length. The WT1/full-length protein was expressed as a fusion protein with Ra12. The WT1/N-terminus protein was expressed as a fusion protein with TRX. The control Ra12 and TRX proteins were purified in a similar manner. The results confirm that the serum antibody reactivity against the WT1 fusion proteins is directed against the WT1 portions of the protein.

FIG. 18 provides the characteristics of the recombinant WT1 proteins used for serological analysis.

FIG. 19A–19E is a bar graph depicting the antibody responses in mice elicited by vaccination with different doses of WT1 protein.

FIGS. 20A and B is a bar graph of the proliferative T-cell responses in mice immunized with WT1 protein.

FIG. 21 is a photograph of human DC, examined by fluorescent microscopy, expressing WT1 following adeno WT1 and Vaccinia WT1 infection.

FIG. 22 is a photograph that demonstrates that WT1 expression in human DC is reproducible following adeno WT1 infection and is not induced by a control Adeno infection.

FIG. 23 is a graph of an IFN-gamma ELISPOT assay showing that WT1 whole gene in vitro priming elicits WT1 specific T-cell responses.

DETAILED DESCRIPTION OF THE INVENTION

As noted above, the present invention is generally directed to compositions and methods for the immunotherapy and diagnosis of malignant diseases. The compositions described herein may include WT1 polypeptides, WT1 polynucleotides, antigen-presenting cells (APC, e.g., dendritic cells) that express a WT1 polypeptide, agents such as antibodies that bind to a WT1 polypeptide and/or immune system cells (e.g., T cells) specific for WT1. WT1 Polypeptides of the present invention generally comprise at least a portion of a Wilms Tumor gene product (WT1) or a variant thereof. Nucleic acid sequences of the subject invention generally comprise a DNA or RNA sequence that encodes all or a portion of such a polypeptide, or that is complementary to such a sequence. Antibodies are generally immune system proteins, or antigen-binding fragments thereof, that are capable of binding to a portion of a WT1 polypeptide. T cells that may be employed within such compositions are generally T cells (e.g., CD4⁺ and/or CD8⁺) that are specific for a WT1 polypeptide. Certain methods described herein further employ antigen-presenting cells that express a WT1 polypeptide as provided herein.

The present invention is based on the discovery that an immune response raised against a Wilms Tumor (WT) gene product (e.g., WT1) can provide prophylactic and/or therapeutic benefit for patients afflicted with malignant diseases characterized by increased WT1 gene expression. Such diseases include, but are not limited to, leukemias (e.g., acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL) and childhood ALL), as well as many cancers such as lung, breast, thyroid and gastrointestinal cancers and melanomas. The WT1 gene was originally identified and isolated on the basis of a cytogenetic deletion at chromosome 11p13 in patients with Wilms' tumor (see Call et al., U.S. Pat. No. 5,350,840). The gene consists of 10 exons and encodes a zinc finger transcription factor, and sequences of mouse and human WT1 proteins are provided in FIG. 1 and SEQ ID NOs: 319 and 320.

WT1 Polypeptides

Within the context of the present invention, a WT1 polypeptide is a polypeptide that comprises at least an immunogenic portion of a native WT1 (i.e., a WT1 protein expressed by an organism that is not genetically modified), or a variant thereof, as described herein. A WT1 polypeptide may be of any length, provided that it comprises at least an immunogenic portion of a native protein or a variant thereof. In other words, a WT1 polypeptide may be an oligopeptide (i.e., consisting of a relatively small number of amino acid residues, such as 8–10 residues, joined by peptide bonds), a full length WT1 protein (e.g., present within a human or non-human animal, such as a mouse) or a polypeptide of intermediate size. Within certain embodiments, the use of WT1 polypeptides that contain a small number of consecutive amino acid residues of a native WT1 polypeptide is preferred. Such polypeptides are preferred for certain uses in which the generation of a T cell response is desired. For example, such a WT1 polypeptide may contain less than 23, preferably no more than 18, and more preferably no more than 15 consecutive amino acid residues, of a native WT1 polypeptide. Polypeptides comprising nine consecutive amino acid residues of a native WT1 polypeptide are generally suitable for such purposes. Additional sequences derived from the native protein and/or heterologous sequences may be present within any WT1 polypeptide, and such sequences may (but need not) possess further immunogenic or antigenic properties. Polypeptides as provided herein may further be associated (covalently or noncovalently) with other polypeptide or non-polypeptide compounds.

An “immunogenic portion,” as used herein is a portion of a polypeptide that is recognized (i.e., specifically bound) by a B-cell and/or T-cell surface antigen receptor. Certain preferred immunogenic portions bind to an MHC class I or class II molecule. As used herein, an immunogenic portion is said to “bind to” an MHC class I or class II molecule if such binding is detectable using any assay known in the art. For example, the ability of a polypeptide to bind to MHC class I may be evaluated indirectly by monitoring the ability to promote incorporation of ¹²⁵I labeled β2-microglobulin (β2m) into MHC class I/β2m/peptide heterotrimeric complexes (see Parker et al., J. Immunol. 152:163, 1994). Alternatively, functional peptide competition assays that are known in the art may be employed. Certain immunogenic portions have one or more of the sequences recited within one or more of Tables II–XIV. Representative immunogenic portions include, but are not limited to, RDLNALLPAVPSLGGGG (human WT1 residues 6–22; SEQ ID NO: 1), PSQASSGQARMFPNAPYLPSCLE (human and mouse WT1 residues 117–139; SEQ ID NOs: 2 and 3 respectively), GATLKGVAAGSSSSVKWTE (human WT1 residues 244–262; SEQ ID NO:4), GATLKGVAA (human WT1 residues 244–252; SEQ ID NO:88), CMTWNQMNL (human and mouse WT1 residues 235–243; SEQ ID NOs: 49 and 258 respectively), SCLESQPTI (mouse WT1 residues 136–144; SEQ ID NO:296), SCLESQPAI (human WT1 residues 136–144; SEQ ID NO:198), NLYQMTSQL (human and mouse WT1 residues 225–233; SEQ ID NOs: 147 and 284 respectively); ALLPAVSSL (mouse WT1 residues 10–18; SEQ ID NO:255); or RMFPNAPYL (human and mouse WT1 residues 126–134; SEQ ID NOs: 185 and 293 respectively). Further immunogenic portions are provided herein, and others may generally be identified using well known techniques, such as those summarized in Paul, Fundamental Immunology, 3rd ed., 243–247 (Raven Press, 1993) and references cited therein. Representative techniques for identifying immunogenic portions include screening polypeptides for the ability to react with antigen-specific antisera and/or T-cell lines or clones. An immunogenic portion of a native WT1 polypeptide is a portion that reacts with such antisera and/or T-cells at a level that is not substantially less than the reactivity of the full length WT1 (e.g., in an ELISA and/or T-cell reactivity assay). In other words, an immunogenic portion may react within such assays at a level that is similar to or greater than the reactivity of the full length polypeptide. Such screens may generally be performed using methods well known to those of ordinary skill in the art, such as those described in Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988.

Alternatively, immunogenic portions may be identified using computer analysis, such as the Tsites program (see Rothbard and Taylor, EMBO J. 7:93–100, 1988; Deavin et al., Mol. Immunol. 33:145–155, 1996), which searches for peptide motifs that have the potential to elicit Th responses. CTL peptides with motifs appropriate for binding to murine and human class I or class II MHC may be identified according to BIMAS (Parker et al., J. Immunol. 152:163, 1994) and other HLA peptide binding prediction analyses. To confirm immunogenicity, a peptide may be tested using an HLA A2 transgenic mouse model and/or an in vitro stimulation assay using dendritic cells, fibroblasts or peripheral blood cells.

As noted above, a composition may comprise a variant of a native WT1 protein. A polypeptide “variant,” as used herein, is a polypeptide that differs from a native polypeptide in one or more substitutions, deletions, additions and/or insertions, such that the immunogenicity of the polypeptide is retained (i.e., the ability of the variant to react with antigen-specific antisera and/or T-cell lines or clones is not substantially diminished relative to the native polypeptide). In other words, the ability of a variant to react with antigen-specific antisera and/or T-cell lines or clones may be enhanced or unchanged, relative to the native polypeptide, or may be diminished by less than 50%, and preferably less than 20%, relative to the native polypeptide. Such variants may generally be identified by modifying one of the above polypeptide sequences and evaluating the reactivity of the modified polypeptide with antisera and/or T-cells as described herein. It has been found, within the context of the present invention, that a relatively small number of substitutions (e.g., 1 to 3) within an immunogenic portion of a WT1 polypeptide may serve to enhance the ability of the polypeptide to elicit an immune response. Suitable substitutions may generally be identified by using computer programs, as described above, and the effect confirmed based on the reactivity of the modified polypeptide with antisera and/or T-cells as described herein. Accordingly, within certain preferred embodiments, a WT1 polypeptide comprises a variant in which 1 to 3 amino acid resides within an immunogenic portion are substituted such that the ability to react with antigen-specific antisera and/or T-cell lines or clones is statistically greater than that for the unmodified polypeptide. Such substitutions are preferably located within an MHC binding site of the polypeptide, which may be identified as described above. Preferred substitutions allow increased binding to MHC class I or class II molecules.

Certain variants contain conservative substitutions. A “conservative substitution” is one in which an amino acid is substituted for another amino acid that has similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic nature of the polypeptide to be substantially unchanged. Amino acid substitutions may generally be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or the amphipathic nature of the residues. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; and serine, threonine, phenylalanine and tyrosine. Other groups of amino acids that may represent conservative changes include: (1) ala, pro, gly, glu, asp, gln, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his. A variant may also, or alternatively, contain nonconservative changes. Variants may also (or alternatively) be modified by, for example, the deletion or addition of amino acids that have minimal influence on the immunogenicity, secondary structure and hydropathic nature of the polypeptide.

In a preferred embodiment, a variant polypeptide of the WT1 N-terminus (amino acids 1–249) is constructed, wherein the variant polypeptide is capable of binding to an antibody that recognizes full-length WT1 and/or WT1 N-terminus polypeptide. A non-limiting example of an antibody is anti WT-1 antibody WT180 (Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.).

As noted above, WT1 polypeptides may be conjugated to a signal (or leader) sequence at the N-terminal end of the protein which co-translationally or post-translationally directs transfer of the protein. A polypeptide may also, or alternatively, be conjugated to a linker or other sequence for ease of synthesis, purification or identification of the polypeptide (e.g., poly-His), or to enhance binding of the polypeptide to a solid support. For example, a polypeptide may be conjugated to an immunoglobulin Fc region.

WT1 polypeptides may be prepared using any of a variety of well known techniques. Recombinant polypeptides encoded by a WT1 polynucleotide as described herein may be readily prepared from the polynucleotide. In general, any of a variety of expression vectors known to those of ordinary skill in the art may be employed to express recombinant WT1 polypeptides. Expression may be achieved in any appropriate host cell that has been transformed or transfected with an expression vector containing a DNA molecule that encodes a recombinant polypeptide. Suitable host cells include prokaryotes, yeast and higher eukaryotic cells. Preferably, the host cells employed are E. coli, yeast or a mammalian cell line such as COS or CHO. Supernatants from suitable host/vector systems which secrete recombinant protein or polypeptide into culture media may be first concentrated using a commercially available filter. The concentrate may then be applied to a suitable purification matrix such as an affinity matrix or an ion exchange resin. Finally, one or more reverse phase HPLC steps can be employed to further purify a recombinant polypeptide. Such techniques may be used to prepare native polypeptides or variants thereof. For example, polynucleotides that encode a variant of a native polypeptide may generally be prepared using standard mutagenesis techniques, such as oligonucleotide-directed site-specific mutagenesis, and sections of the DNA sequence may be removed to permit preparation of truncated polypeptides.

Certain portions and other variants may also be generated by synthetic means, using techniques well known to those of ordinary skill in the art. For example, polypeptides having fewer than about 500 amino acids, preferably fewer than about 100 amino acids, and more preferably fewer than about 50 amino acids, may be synthesized. Polypeptides may be synthesized using any of the commercially available solid-phase techniques, such as the Merrifield solid-phase synthesis method, where amino acids are sequentially added to a growing amino acid chain. See Merrifield, J. Am. Chem. Soc. 85:2149–2146, 1963. Equipment for automated synthesis of polypeptides is commercially available from suppliers such as Applied BioSystems, Inc. (Foster City, Calif.), and may be operated according to the manufacturer's instructions.

In general, polypeptides and polynucleotides as described herein are isolated. An “isolated” polypeptide or polynucleotide is one that is removed from its original environment. For example, a naturally-occurring protein is isolated if it is separated from some or all of the coexisting materials in the natural system. Preferably, such polypeptides are at least about 90% pure, more preferably at least about 95% pure and most preferably at least about 99% pure. A polynucleotide is considered to be isolated if, for example, it is cloned into a vector that is not a part of the natural environment.

Within further aspects, the present invention provides mimetics of WT1 polypeptides. Such mimetics may comprise amino acids linked to one or more amino acid mimetics (i.e., one or more amino acids within the WT1 protein may be replaced by an amino acid mimetic) or may be entirely nonpeptide mimetics. An amino acid mimetic is a compound that is conformationally similar to an amino acid such that it can be substituted for an amino acid within a WT1 polypeptide without substantially diminishing the ability to react with antigen-specific antisera and/or T cell lines or clones. A nonpeptide mimetic is a compound that does not contain amino acids, and that has an overall conformation that is similar to a WT1 polypeptide such that the ability of the mimetic to react with WT1-specific antisera and/or T cell lines or clones is not substantially diminished relative to the ability of a WT1 polypeptide. Such mimetics may be designed based on standard techniques (e.g., nuclear magnetic resonance and computational techniques) that evaluate the three dimensional structure of a peptide sequence. Mimetics may be designed where one or more of the side chain functionalities of the WT1 polypeptide are replaced by groups that do not necessarily have the same size or volume, but have similar chemical and/or physical properties which produce similar biological responses. It should be understood that, within embodiments described herein, a mimetic may be substituted for a WT1 polypeptide.

Within other illustrative embodiments, a polypeptide may be a fusion polypeptide that comprises multiple polypeptides as described herein, or that comprises at least one polypeptide as described herein and an unrelated sequence, such as a known tumor protein. A fusion partner may, for example, assist in providing T helper epitopes (an immunological fusion partner), preferably T helper epitopes recognized by humans, or may assist in expressing the protein (an expression enhancer) at higher yields than the native recombinant protein. Certain preferred fusion partners are both immunological and expression enhancing fusion partners. Other fusion partners may be selected so as to increase the solubility of the polypeptide or to enable the polypeptide to be targeted to desired intracellular compartments. Still further fusion partners include affinity tags, which facilitate purification of the polypeptide.

Fusion polypeptides may generally be prepared using standard techniques, including chemical conjugation. Preferably, a fusion polypeptide is expressed as a recombinant polypeptide, allowing the production of increased levels, relative to a non-fused polypeptide, in an expression system. Briefly, DNA sequences encoding the polypeptide components may be assembled separately, and ligated into an appropriate expression vector. The 3′ end of the DNA sequence encoding one polypeptide component is ligated, with or without a peptide linker, to the 5′ end of a DNA sequence encoding the second polypeptide component so that the reading frames of the sequences are in phase. This permits translation into a single fusion polypeptide that retains the biological activity of both component polypeptides.

A peptide linker sequence may be employed to separate the first and second polypeptide components by a distance sufficient to ensure that each polypeptide folds into its secondary and tertiary structures. Such a peptide linker sequence is incorporated into the fusion polypeptide using standard techniques well known in the art. Suitable peptide linker sequences may be chosen based on the following factors: (1) their ability to adopt a flexible extended conformation; (2) their inability to adopt a secondary structure that could interact with functional epitopes on the first and second polypeptides; and (3) the lack of hydrophobic or charged residues that might react with the polypeptide functional epitopes. Preferred peptide linker sequences contain Gly, Asn and Ser residues. Other near neutral amino acids, such as Thr and Ala may also be used in the linker sequence. Amino acid sequences which may be usefully employed as linkers include those disclosed in Maratea et al., Gene 40:39–46, 1985; Murphy et al., Proc. Natl. Acad. Sci. USA 83:8258–8262, 1986; U.S. Pat. No. 4,935,233 and U.S. Pat. No. 4,751,180. The linker sequence may generally be from 1 to about 50 amino acids in length. Linker sequences are not required when the first and second polypeptides have non-essential N-terminal amino acid regions that can be used to separate the functional domains and prevent steric interference.

The ligated DNA sequences are operably linked to suitable transcriptional or translational regulatory elements. The regulatory elements responsible for expression of DNA are located only 5′ to the DNA sequence encoding the first polypeptides. Similarly, stop codons required to end translation and transcription termination signals are only present 3′ to the DNA sequence encoding the second polypeptide.

The fusion polypeptide can comprise a polypeptide as described herein together with an unrelated immunogenic protein, such as an immunogenic protein capable of eliciting a recall response. Examples of such proteins include tetanus, tuberculosis and hepatitis proteins (see, for example, Stoute et al. New Engl. J. Med., 336:86–91, 1997).

In one preferred embodiment, the immunological fusion partner is derived from a Mycobacterium sp., such as a Mycobacterium tuberculosis-derived Ra12 fragment. Ra12 compositions and methods for their use in enhancing the expression and/or immunogenicity of heterologous polynucleotide/polypeptide sequences is described in U.S. patent application Ser. No. 60/158,585, the disclosure of which is incorporated herein by reference in its entirety. Briefly, Ra12 refers to a polynucleotide region that is a subsequence of a Mycobacterium tuberculosis MTB32A nucleic acid. MTB32A is a serine protease of 32 KD molecular weight encoded by a gene in virulent and avirulent strains of M. tuberculosis. The nucleotide sequence and amino acid sequence of MTB32A have been described (for example, U.S. patent application Ser. No. 60/158,585; see also, Skeiky et al., Infection and Immun. (1999) 67:3998–4007, incorporated herein by reference). C-terminal fragments of the MTB32A coding sequence express at high levels and remain as soluble polypeptides throughout the purification process. Moreover, Ra12 may enhance the immunogenicity of heterologous immunogenic polypeptides with which it is fused. One preferred Ra12 fusion polypeptide comprises a 14 KD C-terminal fragment corresponding to amino acid residues 192 to 323 of MTB32A. Other preferred Ra12 polynucleotides generally comprise at least about 15 consecutive nucleotides, at least about 30 nucleotides, at least about 60 nucleotides, at least about 100 nucleotides, at least about 200 nucleotides, or at least about 300 nucleotides that encode a portion of a Ra12 polypeptide. Ra12 polynucleotides may comprise a native sequence (i.e., an endogenous sequence that encodes a Ra12 polypeptide or a portion thereof) or may comprise a variant of such a sequence. Ra12 polynucleotide variants may contain one or more substitutions, additions, deletions and/or insertions such that the biological activity of the encoded fusion polypeptide is not substantially diminished, relative to a fusion polypeptide comprising a native Ra12 polypeptide. Variants preferably exhibit at least about 70% identity, more preferably at least about 80% identity and most preferably at least about 90% identity to a polynucleotide sequence that encodes a native Ra12 polypeptide or a portion thereof.

Within other preferred embodiments, an immunological fusion partner is derived from protein D, a surface protein of the gram-negative bacterium Haemophilus influenza B (WO 91/18926). Preferably, a protein D derivative comprises approximately the first third of the protein (e.g., the first N-terminal 100–110 amino acids), and a protein D derivative may be lipidated. Within certain preferred embodiments, the first 109 residues of a Lipoprotein D fusion partner is included on the N-terminus to provide the polypeptide with additional exogenous T-cell epitopes and to increase the expression level in E. coli (thus functioning as an expression enhancer). The lipid tail ensures optimal presentation of the antigen to antigen presenting cells. Other fusion partners include the non-structural protein from influenzae virus, NS1 (hemaglutinin). Typically, the N-terminal 81 amino acids are used, although different fragments that include T-helper epitopes may be used.

In another embodiment, the immunological fusion partner is the protein known as LYTA, or a portion thereof (preferably a C-terminal portion). LYTA is derived from Streptococcus pneumoniae, which synthesizes an N-acetyl-L-alanine amidase known as amidase LYTA (encoded by the LytA gene; Gene 43:265–292, 1986). LYTA is an autolysin that specifically degrades certain bonds in the peptidoglyean backbone. The C-terminal domain of the LYTA protein is responsible for the affinity to the choline or to some choline analogues such as DEAE. This property has been exploited for the development of E. coli C-LYTA expressing plasmids useful for expression of fusion proteins. Purification of hybrid proteins containing the C-LYTA fragment at the amino terminus has been described (see Biotechnology 10:795–798, 1992). Within a preferred embodiment, a repeat portion of LYTA may be incorporated into a fusion polypeptide. A repeat portion is found in the C-terminal region starting at residue 178. A particularly preferred repeat portion incorporates residues 188–305.

Yet another illustrative embodiment involves fusion polypeptides, and the polynucleotides encoding them, wherein the fusion partner comprises a targeting signal capable of directing a polypeptide to the endosomal/lysosomal compartment, as described in U.S. Pat. No. 5,633,234. An immunogenic polypeptide of the invention, when fused with this targeting signal, will associate more efficiently with MHC class II molecules and thereby provide enhanced in vivo stimulation of CD4⁺ T-cells specific for the polypeptide.

The invention provides truncated forms of WT-1 polypeptides that can be recombinantly expressed in E. coli without the addition of a fusion partner. Examples of these truncated forms are shown in SEQ ID NOs:342–346, and are encoded by polynucleotides shown in SEQ ID NOs:337–341. In variations of these truncations, the first 76 amino acids of WT-1 can be fused to the C-terminus of the protein, creating a recombinant protein that is easier to express in E. coli. Other hosts in addition to E. coli can also be used, such as, for example, B. megaterium. The protein can further be prepared without a histidine tag.

In other embodiments, different subunits can be made and fused together in an order which differs from that of native WT-1. In addition, fusions can be made with, for example, Ra12. Exemplary fusion proteins are shown in SEQ ID NOs: 332–336 and can be encoded by polynucleotides shown in SEQ ID NOs: 327–331.

WT1 Polynucleotides

Any polynucleotide that encodes a WT1 polypeptide as described herein is a WT1 polynucleotide encompassed by the present invention. Such polynucleotides may be single-stranded (coding or antisense) or double-stranded, and may be DNA (genomic, cDNA or synthetic) or RNA molecules. Additional coding or non-coding sequences may, but need not, be present within a polynucleotide of the present invention, and a polynucleotide may, but need not, be linked to other molecules and/or support materials.

WT1 polynucleotides may encode a native WT1 protein, or may encode a variant of WT1 as described herein. Polynucleotide variants may contain one or more substitutions, additions, deletions and/or insertions such that the immunogenicity of the encoded polypeptide is not diminished, relative to a native WT1 protein. The effect on the immunogenicity of the encoded polypeptide may generally be assessed as described herein. Preferred variants contain nucleotide substitutions, deletions, insertions and/or additions at no more than 20%, preferably at no more than 10%, of the nucleotide positions that encode an immunogenic portion of a native WT1 sequence. Certain variants are substantially homologous to a native gene, or a portion thereof. Such polynucleotide variants are capable of hybridizing under moderately stringent conditions to a naturally occurring DNA sequence encoding a WT1 polypeptide (or a complementary sequence). Suitable moderately stringent conditions include prewashing in a solution of 5×SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); hybridizing at 50° C.–65° C., 5×SSC, overnight; followed by washing twice at 65° C. for 20 minutes with each of 2×, 0.5× and 0.2×SSC containing 0.1% SDS). Such hybridizing DNA sequences are also within the scope of this invention.

It will be appreciated by those of ordinary skill in the art that, as a result of the degeneracy of the genetic code, there are many nucleotide sequences that encode a WT1 polypeptide. Some of these polynucleotides bear minimal homology to the nucleotide sequence of any native gene. Nonetheless, polynucleotides that vary due to differences in codon usage are specifically contemplated by the present invention.

Once an immunogenic portion of WT1 is identified, as described above, a WT1 polynucleotide may be prepared using any of a variety of techniques. For example, a WT1 polynucleotide may be amplified from cDNA prepared from cells that express WT1. Such polynucleotides may be amplified via polymerase chain reaction (PCR). For this approach, sequence-specific primers may be designed based on the sequence of the immunogenic portion and may be purchased or synthesized. For example, suitable primers for PCR amplification of a human WT1 gene include: first step—P118: 1434–1414: 5′ GAG AGT CAG ACT TGA AAG CAGT 3′ (SEQ ID NO:5) and P135: 5° CTG AGC CTC AGC AAA TGG GC 3′ (SEQ ID NO:6); second step—P136: 5′ GAG CAT GCA TGG GCT CCG ACG TGC GGG 3′ (SEQ ID NO:7) and P137: 5′ GGG GTA CCC ACT GAA CGG TCC CCG A 3′ (SEQ ID NO:8). Primers for PCR amplification of a mouse WT1 gene include: first step—P138: 5′ TCC GAG CCG CAC CTC ATG 3′ (SEQ ID NO:9) and P139: 5′ GCC TGG GAT GCT GGA CTG 3′ (SEQ ID NO:10), second step—P140: 5′ GAG CAT GCG ATG GGT TCC GAC GTG CGG 3′ (SEQ ID NO:11) and P141: 5′ GGG GTA CCT CAA AGC GCC ACG TGG AGT TT 3′ (SEQ ID NO:12).

An amplified portion may then be used to isolate a full length gene from a human genomic DNA library or from a suitable cDNA library, using well known techniques. Alternatively, a full length gene can be constructed from multiple PCR fragments. WT1 polynucleotides may also be prepared by synthesizing oligonucleotide components, and ligating components together to generate the complete polynucleotide.

WT1 polynucleotides may also be synthesized by any method known in the art, including chemical synthesis (e.g., solid phase phosphoramidite chemical synthesis). Modifications in a polynucleotide sequence may also be introduced using standard mutagenesis techniques, such as oligonucleotide-directed site-specific mutagenesis (see Adelman et al., DNA 2:183, 1983). Alternatively, RNA molecules may be generated by in vitro or in vivo transcription of DNA sequences encoding a WT1 polypeptide, provided that the DNA is incorporated into a vector with a suitable RNA polymerase promoter (such as T7 or SP6). Certain portions may be used to prepare an encoded polypeptide, as described herein. In addition, or alternatively, a portion may be administered to a patient such that the encoded polypeptide is generated in vivo (e.g., by transfecting antigen-presenting cells such as dendritic cells with a cDNA construct encoding a WT1 polypeptide, and administering the transfected cells to the patient).

Polynucleotides that encode a WT1 polypeptide may generally be used for production of the polypeptide, in vitro or in vivo. WT1 polynucleotides that are complementary to a coding sequence (i.e., antisense polynucleotides) may also be used as a probe or to inhibit WT1 expression. cDNA constructs that can be transcribed into antisense RNA may also be introduced into cells of tissues to facilitate the production of antisense RNA.

Any polynucleotide may be further modified to increase stability in vivo. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5′ and/or 3′ ends; the use of phosphorothioate or 2′O-methyl rather than phosphodiesterase linkages in the backbone; and/or the inclusion of nontraditional bases such as inosine, queosine and wybutosine, as well as acetyl-methyl-, thio- and other modified forms of adenine, cytidine, guanine, thymine and uridine.

Nucleotide sequences as described herein may be joined to a variety of other nucleotide sequences using established recombinant DNA techniques. For example, a polynucleotide may be cloned into any of a variety of cloning vectors, including plasmids, phagemids, lambda phage derivatives and cosmids. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors and sequencing vectors. In general, a vector will contain an origin of replication functional in at least one organism, convenient restriction endonuclease sites and one or more selectable markers. Other elements will depend upon the desired use, and will be apparent to those of ordinary skill in the art.

Within certain embodiments, polynucleotides may be formulated so as to permit entry into a cell of a mammal, and expression therein. Such formulations are particularly useful for therapeutic purposes, as described below. Those of ordinary skill in the art will appreciate that there are many ways to achieve expression of a polynucleotide in a target cell, and any suitable method may be employed. For example, a polynucleotide may be incorporated into a viral vector such as, but not limited to, adenovirus, adeno-associated virus, retrovirus, or vaccinia or other pox virus (e.g., avian pox virus). Techniques for incorporating DNA into such vectors are well known to those of ordinary skill in the art. A retroviral vector may additionally transfer or incorporate a gene for a selectable marker (to aid in the identification or selection of transduced cells) and/or a targeting moiety, such as a gene that encodes a ligand for a receptor on a specific target cell, to render the vector target specific. Targeting may also be accomplished using an antibody, by methods known to those of ordinary skill in the art. cDNA constructs within such a vector may be used, for example, to transfect human or animal cell lines for use in establishing WT1 positive tumor models which may be used to perform tumor protection and adoptive immunotherapy experiments to demonstrate tumor or leukemia-growth inhibition or lysis of such cells.

Other therapeutic formulations for polynucleotides include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. A preferred colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (i.e., an artificial membrane vesicle). The preparation and use of such systems is well known in the art.

Antibodies and Fragments Thereof

The present invention further provides binding agents, such as antibodies and antigen-binding fragments thereof, that specifically bind to a WT1 polypeptide. As used herein, an agent is said to “specifically bind” to a WT1 polypeptide if it reacts at a detectable level (within, for example, an ELISA) with a WT1 polypeptide, and does not react detectably with unrelated proteins under similar conditions. As used herein, “binding” refers to a noncovalent association between two separate molecules such that a “complex” is formed. The ability to bind may be evaluated by, for example, determining a binding constant for the formation of the complex. The binding constant is the value obtained when the concentration of the complex is divided by the product of the component concentrations. In general, two compounds are said to “bind,” in the context of the present invention, when the binding constant for complex formation exceeds about 10³ L/mol. The binding constant maybe determined using methods well known in the art.

Any agent that satisfies the above requirements may be a binding agent. In a preferred embodiment, a binding agent is an antibody or an antigen-binding fragment thereof. Certain antibodies are commercially available from, for example, Santa Cruz Biotechnology (Santa Cruz, Calif.). Alternatively, antibodies may be prepared by any of a variety of techniques known to those of ordinary skill in the art. See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. In general, antibodies can be produced by cell culture techniques, including the generation of monoclonal antibodies as described herein, or via transfection of antibody genes into suitable bacterial or mammalian cell hosts, in order to allow for the production of recombinant antibodies. In one technique, an immunogen comprising the polypeptide is initially injected into any of a wide variety of mammals (e.g., mice, rats, rabbits, sheep or goats). In this step, the polypeptides of this invention may serve as the immunogen without modification. Alternatively, particularly for relatively short polypeptides, a superior immune response may be elicited if the polypeptide is joined to a carrier protein, such as bovine serum albumin or keyhole limpet hemocyanin. The immunogen is injected into the animal host, preferably according to a predetermined schedule incorporating one or more booster immunizations, and the animals are bled periodically. Polyclonal antibodies specific for the polypeptide may then be purified from such antisera by, for example, affinity chromatography using the polypeptide coupled to a suitable solid support.

Monoclonal antibodies specific for the antigenic polypeptide of interest may be prepared, for example, using the technique of Kohler and Milstein, Eur. J. Immunol 6:511–519, 1976, and improvements thereto. Briefly, these methods involve the preparation of immortal cell lines capable of producing antibodies having the desired specificity (i.e., reactivity with the polypeptide of interest). Such cell lines may be produced, for example, from spleen cells obtained from an animal immunized as described above. The spleen cells are then immortalized by, for example, fusion with a myeloma cell fusion partner, preferably one that is syngeneic with the immunized animal. A variety of fusion techniques may be employed. For example, the spleen cells and myeloma cells may be combined with a nonionic detergent for a few minutes and then plated at low density on a selective medium that supports the growth of hybrid cells, but not myeloma cells. A preferred selection technique uses HAT (hypoxanthine, aminopterin, thymidine) selection. After a sufficient time, usually about 1 to 2 weeks, colonies of hybrids are observed. Single colonies are selected and their culture supernatants tested for binding activity against the polypeptide. Hybridomas having high reactivity and specificity are preferred.

Monoclonal antibodies may be isolated from the supernatants of growing hybridoma colonies. In addition, various techniques may be employed to enhance the yield, such as injection of the hybridoma cell line into the peritoneal cavity of a suitable vertebrate host, such as a mouse. Monoclonal antibodies may then be harvested from the ascites fluid or the blood. Contaminants may be removed from the antibodies by conventional techniques, such as chromatography, gel filtration, precipitation, and extraction. The polypeptides of this invention may be used in the purification process in, for example, an affinity chromatography step.

Within certain embodiments, the use of antigen-binding fragments of antibodies may be preferred. Such fragments include Fab fragments, which may be prepared using standard techniques. Briefly, immunoglobulins may be purified from rabbit serum by affinity chromatography on Protein A bead columns (Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988) and digested by papain to yield Fab and Fc fragments. The Fab and Fc fragments may be separated by affinity chromatography on protein A bead columns.

Monoclonal antibodies and fragments thereof may be coupled to one or more therapeutic agents. Suitable agents in this regard include radioactive tracers and chemotherapeutic agents, which may be used, for example, to purge autologous bone marrow in vitro). Representative therapeutic agents include radionuclides, differentiation inducers, drugs, toxins, and derivatives thereof. Preferred radionuclides include ⁹⁰Y, ¹²³I, ¹²⁵I, ¹³¹I, ¹⁸⁶Re, ¹⁸⁸Re, ²¹¹At, and ²¹²Bi. Preferred drugs include methotrexate, and pyrimidine and purine analogs. Preferred differentiation inducers include phorbol esters and butyric acid. Preferred toxins include ricin, abrin, diptheria toxin, cholera toxin, gelonin, Pseudomonas exotoxin, Shigella toxin, and pokeweed antiviral protein. For diagnostic purposes, coupling of radioactive agents may be used to facilitate tracing of metastases or to determine the location of WT1-positive tumors.

A therapeutic agent may be coupled (e.g., covalently bonded) to a suitable monoclonal antibody either directly or indirectly (e.g., via a linker group). A direct reaction between an agent and an antibody is possible when each possesses a substituent capable of reacting with the other. For example, a nucleophilic group, such as an amino or sulfhydryl group, on one may be capable of reacting with a carbonyl-containing group, such as an anhydride or an acid halide, or with an alkyl group containing a good leaving group (e.g., a halide) on the other.

Alternatively, it may be desirable to couple a therapeutic agent and an antibody via a linker group. A linker group can function as a spacer to distance an antibody from an agent in order to avoid interference with binding capabilities. A linker group can also serve to increase the chemical reactivity of a substituent on an agent or an antibody, and thus increase the coupling efficiency. An increase in chemical reactivity may also facilitate the use of agents, or functional groups on agents, which otherwise would not be possible.

It will be evident to those skilled in the art that a variety of bifunctional or polyfunctional reagents, both homo- and hetero-functional (such as those described in the catalog of the Pierce Chemical Co., Rockford, Ill.), may be employed as the linker group. Coupling may be effected, for example, through amino groups, carboxyl groups, sulfhydryl groups or oxidized carbohydrate residues. There are numerous references describing such methodology, e.g., U.S. Pat. No. 4,671,958, to Rodwell et al.

Where a therapeutic agent is more potent when free from the antibody portion of the immunoconjugates of the present invention, it may be desirable to use a linker group which is cleavable during or upon internalization into a cell. A number of different cleavable linker groups have been described. The mechanisms for the intracellular release of an agent from these linker groups include cleavage by reduction of a disulfide bond (e.g., U.S. Pat. No. 4,489,710, to Spitler), by irradiation of a photolabile bond (e.g., U.S. Pat. No. 4,625,014, to Senter et al.), by hydrolysis of derivatized amino acid side chains (e.g., U.S. Pat. No. 4,638,045, to Kohn et al.), by serum complement-mediated hydrolysis (e.g., U.S. Pat. No. 4,671,958, to Rodwell et al.), and acid-catalyzed hydrolysis (e.g., U.S. Pat. No. 4,569,789, to Blattler et al.).

It may be desirable to couple more than one agent to an antibody. In one embodiment, multiple molecules of an agent are coupled to one antibody molecule. In another embodiment, more than one type of agent may be coupled to one antibody. Regardless of the particular embodiment, immunoconjugates with more than one agent may be prepared in a variety of ways. For example, more than one agent may be coupled directly to an antibody molecule, or linkers which provide multiple sites for attachment can be used. Alternatively, a carrier can be used. A carrier may bear the agents in a variety of ways, including covalent bonding either directly or via a linker group. Suitable carriers include proteins such as albumins (e.g., U.S. Pat. No. 4,507,234, to Kato et al.), peptides and polysaccharides such as aminodextran (e.g., U.S. Pat. No. 4,699,784, to Shih et al.). A carrier may also bear an agent by noncovalent bonding or by encapsulation, such as within a liposome vesicle (e.g., U.S. Pat. Nos. 4,429,008 and 4,873,088). Carriers specific for radionuclide agents include radiohalogenated small molecules and chelating compounds. For example, U.S. Pat. No. 4,735,792 discloses representative radiohalogenated small molecules and their synthesis. A radionuclide chelate may be formed from chelating compounds that include those containing nitrogen and sulfur atoms as the donor atoms for binding the metal, or metal oxide, radionuclide. For example, U.S. Pat. No. 4,673,562, to Davison et al. discloses representative chelating compounds and their synthesis.

A variety of routes of administration for the antibodies and immunoconjugates may be used. Typically, administration will be intravenous, intramuscular, subcutaneous or in the bed of a resected tumor. It will be evident that the precise dose of the antibody/immunoconjugate will vary depending upon the antibody used, the antigen density on the tumor, and the rate of clearance of the antibody.

Also provided herein are anti-idiotypic antibodies that mimic an immunogenic portion of WT1. Such antibodies may be raised against an antibody, or antigen-binding fragment thereof, that specifically binds to an immunogenic portion of WT1, using well known techniques. Anti-idiotypic antibodies that mimic an immunogenic portion of WT1 are those antibodies that bind to an antibody, or antigen-binding fragment thereof, that specifically binds to an immunogenic portion of WT1, as described herein.

T Cells

Immunotherapeutic compositions may also, or alternatively, comprise T cells specific for WT1. Such cells may generally be prepared in vitro or ex vivo, using standard procedures. For example, T cells may be present within (or isolated from) bone marrow, peripheral blood or a fraction of bone marrow or peripheral blood of a mammal, such as a patient, using a commercially available cell separation system, such as the CEPRATE™ system, available from CellPro Inc., Bothell, Wash. (see also U.S. Pat. No. 5,240,856; U.S. Pat. No. 5,215,926; WO 89/06280; WO 91/16116 and WO 92/07243). Alternatively, T cells may be derived from related or unrelated humans, non-human animals, cell lines or cultures.

T cells may be stimulated with WT1 polypeptide, polynucleotide encoding a WT1 polypeptide and/or an antigen presenting cell (APC) that expresses a WT1 polypeptide. Such stimulation is performed under conditions and for a time sufficient to permit the generation of T cells that are specific for the WT1 polypeptide. Preferably, a WT1 polypeptide or polynucleotide is present within a delivery vehicle, such as a microsphere, to facilitate the generation of antigen-specific T cells. Briefly, T cells, which may be isolated from a patient or a related or unrelated donor by routine techniques (such as by Ficoll/Hypaque density gradient centrifugation of peripheral blood lymphocytes), are incubated with WT1 polypeptide. For example, T cells may be incubated in vitro for 2–9 days (typically 4 days) at 37° C. with WT1 polypeptide (e.g., 5 to 25 μg/ml) or cells synthesizing a comparable amount of WT1 polypeptide. It may be desirable to incubate a separate aliquot of a T cell sample in the absence of WT1 polypeptide to serve as a control.

T cells are considered to be specific for a WT1 polypeptide if the T cells kill target cells coated with a WT1 polypeptide or expressing a gene encoding such a polypeptide. T cell specificity may be evaluated using any of a variety of standard techniques. For example, within a chromium release assay or proliferation assay, a stimulation index of more than two fold increase in lysis and/or proliferation, compared to negative controls, indicates T cell specificity. Such assays may be performed, for example, as described in Chen et al., Cancer Res. 54:1065–1070, 1994. Alternatively, detection of the proliferation of T cells may be accomplished by a variety of known techniques. For example, T cell proliferation can be detected by measuring an increased rate of DNA synthesis (e.g., by pulse-labeling cultures of T cells with tritiated thymidine and measuring the amount of tritiated thymidine incorporated into DNA). Other ways to detect T cell proliferation include measuring increases in interleukin-2 (IL-2) production, Ca²⁺ flux, or dye uptake, such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium. Alternatively, synthesis of lymphokines (such as interferon-gamma) can be measured or the relative number of T cells that can respond to a WT1 polypeptide may be quantified. Contact with a WT1 polypeptide (200 ng/ml–100 μg/ml, preferably 100 ng/ml–25 μg/ml) for 3–7 days should result in at least a two fold increase in proliferation of the T cells and/or contact as described above for 2–3 hours should result in activation of the T cells, as measured using standard cytokine assays in which a two fold increase in the level of cytokine release (e.g., TNF or IFN-γ) is indicative of T cell activation (see Coligan et al., Current Protocols in Immunology, vol. 1, Wiley Interscience (Greene 1998). WT1 specific T cells may be expanded using standard techniques. Within preferred embodiments, the T cells are derived from a patient or a related or unrelated donor and are administered to the patient following stimulation and expansion.

T cells that have been activated in response to a WT1 polypeptide, polynucleotide or WT1-expressing APC may be CD4⁺ and/or CD8⁺. Specific activation of CD4⁺ or CD8⁺ T cells may be detected in a variety of ways. Methods for detecting specific T cell activation include detecting the proliferation of T cells, the production of cytokines (e.g., lymphokines), or the generation of cytolytic activity (i.e., generation of cytotoxic T cells specific for WT1). For CD4⁺ T cells, a preferred method for detecting specific T cell activation is the detection of the proliferation of T cells. For CD8⁺ T cells, a preferred method for detecting specific T cell activation is the detection of the generation of cytolytic activity.

For therapeutic purposes, CD4⁺ or CD8⁺ T cells that proliferate in response to the WT1 polypeptide, polynucleotide or APC can be expanded in number either in vitro or in vivo. Proliferation of such T cells in vitro may be accomplished in a variety of ways. For example, the T cells can be re-exposed to WT1 polypeptide, with or without the addition of T cell growth factors, such as interleukin-2, and/or stimulator cells that synthesize a WT1 polypeptide. The addition of stimulator cells is preferred where generating CD8⁺ T cell responses. T cells can be grown to large numbers in vitro with retention of specificity in response to intermittent restimulation with WT1 polypeptide. Briefly, for the primary in vitro stimulation (IVS), large numbers of lymphocytes (e.g., greater than 4×10⁷) may be placed in flasks with media containing human serum. WT1 polypeptide (e.g., peptide at 10 μg/ml) may be added directly, along with tetanus toxoid (e.g., 5 μg/ml). The flasks may then be incubated (e.g., 37° C. for 7 days). For a second IVS, T cells are then harvested and placed in new flasks with 2–3×10⁷ irradiated peripheral blood mononuclear cells. WT1 polypeptide (e.g., 10 μg/ml) is added directly. The flasks are incubated at 37° C. for 7 days. On day 2 and day 4 after the second IVS, 2–5 units of interleukin-2 (IL-2) may be added. For a third IVS, the T cells may be placed in wells and stimulated with the individual's own EBV transformed B cells coated with the peptide. IL-2 may be added on days 2 and 4 of each cycle. As soon as the cells are shown to be specific cytotoxic T cells, they may be expanded using a 10 day stimulation cycle with higher IL-2 (20 units) on days 2, 4 and 6.

Alternatively, one or more T cells that proliferate in the presence of WT1 polypeptide can be expanded in number by cloning. Methods for cloning cells are well known in the art, and include limiting dilution. Responder T cells may be purified from the peripheral blood of sensitized patients by density gradient centrifugation and sheep red cell resetting and established in culture by stimulating with the nominal antigen in the presence of irradiated autologous filler cells. In order to generate CD4⁺ T cell lines, WT1 polypeptide is used as the antigenic stimulus and autologous peripheral blood lymphocytes (PBL) or lymphoblastoid cell lines (LCL) immortalized by infection with Epstein Barr virus are used as antigen presenting cells. In order to generate CD8⁺ T cell lines, autologous antigen-presenting cells transfected with an expression vector which produces WT1 polypeptide may be used as stimulator cells. Established T cell lines may be cloned 2–4 days following antigen stimulation by plating stimulated T cells at a frequency of 0.5 cells per well in 96-well flat-bottom plates with 1×10⁶ irradiated PBL or LCL cells and recombinant interleukin-2 (rIL2) (50 U/ml). Wells with established clonal growth may be identified at approximately 2–3 weeks after initial plating and restimulated with appropriate antigen in the presence of autologous antigen-presenting cells, then subsequently expanded by the addition of low doses of rIL2 (10 U/ml) 2–3 days following antigen stimulation. T cell clones may be maintained in 24-well plates by periodic restimulation with antigen and rIL2 approximately every two weeks.

Within certain embodiments, allogeneic T-cells may be primed (i.e., sensitized to WT1) in vivo and/or in vitro. Such priming may be achieved by contacting T cells with a WT1 polypeptide, a polynucleotide encoding such a polypeptide or a cell producing such a polypeptide under conditions and for a time sufficient to permit the priming of T cells. In general, T cells are considered to be primed if, for example, contact with a WT1 polypeptide results in proliferation and/or activation of the T cells, as measured by standard proliferation, chromium release and/or cytokine release assays as described herein. A stimulation index of more than two fold increase in proliferation or lysis, and more than three fold increase in the level of cytokine, compared to negative controls, indicates T-cell specificity. Cells primed in vitro may be employed, for example, within a bone marrow transplantation or as donor lymphocyte infusion.

T cells specific for WT1 can kill cells that express WT1 protein. Introduction of genes encoding T-cell receptor (TCR) chains for WT1 are used as a means to quantitatively and qualitatively improve responses to WT1 bearing leukemia and cancer cells. Vaccines to increase the number of T cells that can react to WT1 positive cells are one method of targeting WT1 bearing cells. T cell therapy with T cells specific for WT1 is another method. An alternative method is to introduce the TCR chains specific for WT1 into T cells or other cells with lytic potential. In a suitable embodiment, the TCR alpha and beta chains are cloned out from a WT1 specific T cell line and used for adoptive T cell therapy, such as described in WO96/30516, incorporated herein by reference.

Pharmaceutical Compositions and Vaccines

Within certain aspects, polypeptides, polynucleotides, antibodies and/or T cells may be incorporated into pharmaceutical compositions or vaccines. Alternatively, a pharmaceutical composition may comprise an antigen-presenting cell (e.g., a dendritic cell) transfected with a WT1 polynucleotide such that the antigen presenting cell expresses a WT1 polypeptide. Pharmaceutical compositions comprise one or more such compounds or cells and a physiologically acceptable carrier or excipient. Certain vaccines may comprise one or more such compounds or cells and a non-specific immune response enhancer, such as an adjuvant or a liposome (into which the compound is incorporated). Pharmaceutical compositions and vaccines may additionally contain a delivery system, such as biodegradable microspheres which are disclosed, for example, in U.S. Pat. Nos. 4,897,268 and 5,075,109. Pharmaceutical compositions and vaccines within the scope of the present invention may also contain other compounds, which may be biologically active or inactive.

Within certain embodiments, pharmaceutical compositions and vaccines are designed to elicit T cell responses specific for a WT1 polypeptide in a patient, such as a human. In general, T cell responses may be favored through the use of relatively short polypeptides (e.g., comprising less than 23 consecutive amino acid residues of a native WT1 polypeptide, preferably 4–16 consecutive residues, more preferably 8–16 consecutive residues and still more preferably 8–10 consecutive residues. Alternatively, or in addition, a vaccine may comprise a non-specific immune response enhancer that preferentially enhances a T cell response. In other words, the immune response enhancer may enhance the level of a T cell response to a WT1 polypeptide by an amount that is proportionally greater than the amount by which an antibody response is enhanced. For example, when compared to a standard oil based adjuvant, such as CFA, an immune response enhancer that preferentially enhances a T cell response may enhance a proliferative T cell response by at least two fold, a lytic response by at least 10%, and/or T cell activation by at least two fold compared to WT1-megative control cell lines, while not detectably enhancing an antibody response. The amount by which a T cell or antibody response to a WT1 polypeptide is enhanced may generally be determined using any representative technique known in the art, such as the techniques provided herein.

A pharmaceutical composition or vaccine may contain DNA encoding one or more of the polypeptides as described above, such that the polypeptide is generated in situ. As noted above, the DNA may be present within any of a variety of delivery systems known to those of ordinary skill in the art, including nucleic acid expression systems, bacterial and viral expression systems and mammalian expression systems. Appropriate nucleic acid expression systems contain the necessary DNA, cDNA or RNA sequences for expression in the patient (such as a suitable promoter and terminating signal). Bacterial delivery systems involve the administration of a bacterium (such as Bacillus-Calmette-Guerrin) that expresses an immunogenic portion of the polypeptide on its cell surface. In a preferred embodiment, the DNA may be introduced using a viral expression system (e.g., vaccinia or other pox virus, retrovirus, or adenovirus), which may involve the use of a non-pathogenic (defective), replication competent virus. Techniques for incorporating DNA into such expression systems are well known to those of ordinary skill in the art. The DNA may also be “naked,” as described, for example, in Ulmer et al., Science 259:1745–1749, 1993 and reviewed by Cohen, Science 259:1691–1692, 1993. The uptake of naked DNA may be increased by coating the DNA onto biodegradable beads, which are efficiently transported into the cells.

As noted above, a pharmaceutical composition or vaccine may comprise an antigen-presenting cell that expresses a WT1 polypeptide. For therapeutic purposes, as described herein, the antigen presenting cell is preferably an autologous dendritic cell. Such cells may be prepared and transfected using standard techniques, such as those described by Reeves et al., Cancer Res. 56:5672–5677, 1996; Tuting et al., J. Immunol. 160:1139–1147, 1998; and Nair et al., Nature Biotechnol. 16:364–369, 1998). Expression of a WT1 polypeptide on the surface of an antigen-presenting cell may be confirmed by in vitro stimulation and standard proliferation as well as chromium release assays, as described herein.

While any suitable carrier known to those of ordinary skill in the art may be employed in the pharmaceutical compositions of this invention, the type of carrier will vary depending on the mode of administration. Compositions of the present invention may be formulated for any appropriate manner of administration, including for example, topical, oral, nasal, intravenous, intracranial, intraperitoneal, subcutaneous or intramuscular administration. For parenteral administration, such as subcutaneous injection, the carrier preferably comprises water, saline, alcohol, a fat, a wax or a buffer. For oral administration, any of the above carriers or a solid carrier, such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, sucrose, and magnesium carbonate, may be employed. Biodegradable microspheres (e.g., polylactate polyglycolate) may also be employed as carriers for the pharmaceutical compositions of this invention. For certain topical applications, formulation as a cream or lotion, using well known components, is preferred.

Such compositions may also comprise buffers (e.g., neutral buffered saline or phosphate buffered saline), carbohydrates (e.g., glucose, mannose, sucrose or dextrans), mannitol, proteins, polypeptides or amino acids such as glycine, antioxidants, chelating agents such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide) and/or preservatives. Alternatively, compositions of the present invention may be formulated as a lyophilizate. Compounds may also be encapsulated within liposomes using well known technology.

Any of a variety of non-specific immune response enhancers, such as adjuvants, may be employed in the vaccines of this invention. Most adjuvants contain a substance designed to protect the antigen from rapid catabolism, such as aluminum hydroxide or mineral oil, and a stimulator of immune responses, such as lipid A, Bortadella pertussis or Mycobacterium tuberculosis derived proteins. Suitable non-specific immune response enhancers include alum-based adjuvants (e.g., Alhydrogel, Rehydragel, aluminum phosphate, Algammulin, aluminum hydroxide); oil based adjuvants (Freund's adjuvant (FA), Specol, RIBI, TiterMax, Montanide ISA50 or Seppic MONTANIDE ISA 720; cytokines (e.g., GM-CSF or Flat3-ligand); microspheres; nonionic block copolymer-based adjuvants; dimethyl dioctadecyl ammoniumbromide (DDA) based adjuvants AS-1, AS-2 (Smith Kline Beecham); Ribi Adjuvant system based adjuvants; QS21 (Aquila); saponin based adjuvants (crude saponin, the saponin Quil A ); muramyl dipeptide (MDP) based adjuvants such as SAF (Syntex adjuvant in its microfluidized form (SAF-m)); dimethyl-dioctadecyl ammonium bromide (DDA); human complement based adjuvants m. vaccae and derivatives; immune stimulating complex (iscom) based adjuvants; inactivated toxins; and attenuated infectious agents (such as M. tuberculosis).

As noted above, within certain embodiments, immune response enhancers are chosen for their ability to preferentially elicit or enhance a T cell response (e.g., CD4⁺ and/or CD8⁺) to a WT1 polypeptide. Such immune response enhancers are well known in the art, and include (but are not limited to) Montanide ISA50, Seppic MONTANIDE ISA 720, cytokines (e.g., GM-CSF, Flat3-ligand), microspheres, dimethyl dioctadecyl ammoniumbromide (DDA) based adjuvants, AS-1 (Smith Kline Beecham), AS-2 (Smith Kline Beecham), Ribi Adjuvant system based adjuvants, QS21 (Aquila), saponin based adjuvants (crude saponin, the saponin Quil A), Syntex adjuvant in its microfluidized form (SAF-m), MV, ddMV (Genesis), immune stimulating complex (iscom) based adjuvants and inactivated toxins.

The compositions and vaccines described herein may be administered as part of a sustained release formulation (i.e., a formulation such as a capsule or sponge that effects a slow release of compound following administration). Such formulations may generally be prepared using well known technology and administered by, for example, oral, rectal or subcutaneous implantation, or by implantation at the desired target site. Sustained-release formulations may contain a polypeptide, polynucleotide, antibody or cell dispersed in a carrier matrix and/or contained within a reservoir surrounded by a rate controlling membrane. Carriers for use within such formulations are biocompatible, and may also be biodegradable; preferably the formulation provides a relatively constant level of active component release. The amount of active compound contained within a sustained release formulation depends upon the site of implantation, the rate and expected duration of release and the nature of the condition to be treated or prevented.

Therapy of Malignant Diseases

In further aspects of the present invention, the compositions and vaccines described herein may be used to inhibit the development of malignant diseases (e.g., progressive or metastatic diseases or diseases characterized by small tumor burden such as minimal residual disease). In general, such methods may be used to prevent, delay or treat a disease associated with WT1 expression. In other words, therapeutic methods provided herein may be used to treat an existing WT1-associated disease, or may be used to prevent or delay the onset of such a disease in a patient who is free of disease or who is afflicted with a disease that is not yet associated with WT1 expression.

As used herein, a disease is “associated with WT1 expression” if diseased cells (e.g., tumor cells) at some time during the course of the disease generate detectably higher levels of a WT1 polypeptide than normal cells of the same tissue. Association of WT1 expression with a malignant disease does not require that WT1 be present on a tumor. For example, overexpression of WT1 may be involved with initiation of a tumor, but the protein expression may subsequently be lost. Alternatively, a malignant disease that is not characterized by an increase in WT1 expression may, at a later time, progress to a disease that is characterized by increased WT1 expression. Accordingly, any malignant disease in which diseased cells formerly expressed, currently express or are expected to subsequently express increased levels of WT1 is considered to be “associated with WT1 expression.”

Immunotherapy may be performed using any of a variety of techniques, in which compounds or cells provided herein function to remove WT1-expressing cells from a patient. Such removal may take place as a result of enhancing or inducing an immune response in a patient specific for WT1 or a cell expressing WT1. Alternatively, WT1-expressing cells may be removed ex vivo (e.g., by treatment of autologous bone marrow, peripheral blood or a fraction of bone marrow or peripheral blood). Fractions of bone marrow or peripheral blood may be obtained using any standard technique in the art.

Within such methods, pharmaceutical compositions and vaccines may be administered to a patient. As used herein, a “patient” refers to any warm-blooded animal, preferably a human. A patient may or may not be afflicted with a malignant disease. Accordingly, the above pharmaceutical compositions and vaccines may be used to prevent the onset of a disease (i.e., prophylactically) or to treat a patient afflicted with a disease (e.g., to prevent or delay progression and/or metastasis of an existing disease). A patient afflicted with a disease may have a minimal residual disease (e.g., a low tumor burden in a leukemia patient in complete or partial remission or a cancer patient following reduction of the tumor burden after surgery radiotherapy and/or chemotherapy). Such a patient may be immunized to inhibit a relapse (i.e., prevent or delay the relapse, or decrease the severity of a relapse). Within certain preferred embodiments, the patient is afflicted with a leukemia (e.g., AML, CML, ALL or childhood ALL), a myelodysplastic syndrome (MDS) or a cancer (e.g., gastrointestinal, lung, thyroid or breast cancer or a melanoma), where the cancer or leukemia is WT1 positive (i.e., reacts detectably with an anti-WT1 antibody, as provided herein or expresses WT1 mRNA at a level detectable by RT-PCR, as described herein) or suffers from an autoimmune disease directed against WT1-expressing cells.

Other diseases associated with WT1 overexpression include kidney cancer (such as renal cell carcinoma, or Wilms tumor), as described in Satoh F., et al., Pathol. Int. 50(6):458–71(2000), and Campbell C. E. et al., Int. J. Cancer 78(2):182–8 (1998) mesothelioma, as described in Amin, K. M. et al., Am. J. Pathol. 146(2):344–56 (1995). Harada et al. (Mol. Urol. 3(4):357–364 (1999) describe WT1 gene expression in human testicular germ-cell tumors. Nonomura et al. Hinyokika Kiyo 45(8):593–7 (1999) describe molecular staging of testicular cancer using polymerase chain reaction of the testicular cancer-specific genes. Shimizu et al., Int. J. Gynecol. Pathol. 19(2):158–63 (2000) describe the immunohistochemical detection of the Wilms' tumor gene (WT1) in epithelial ovarian tumors.

WT1 overexpression was also described in desmoplastic small round cell tumors, by Bamoud, R. et al., Am. J. Surg. Pathol. 24(6):830–6 (2000); and Pathol. Res. Pract. 194(10):693–700 (1998). WT1 overexpression in glioblastoma and other cancer was described by Menssen, H. D. et al., J. Cancer Res. Clin. Oncol. 126(4):226–32 (2000), “Wilms' tumor gene (WT1) expression in lung cancer, colon cancer and glioblastoma cell lines compared to freshly isolated tumor specimens.” Other diseases showing WT1 overexpression include EBV associated diseases, such as Burkitt's lymphoma and nasopharyngeal cancer (Spinsanti P. et al., Leuk. Lymphoma 38(5–6):611–9 (2000), “Wilms' tumor gene expression by normal and malignant human B lymphocytes.”

In Leukemia 14(9):1634–4 (2000), Pan et al., describe in vitro IL-12 treatment of peripheral blood mononuclear cells from patients with leukemia or myelodysplastic syndromes, and reported an increase in cytotoxicity and reduction in WT1 gene expression. In Leukemia 13(6):891–900 (1999), Patmasiriwat et al. reported WT1 and GATA1 expression in myelodysplastic syndrome and acute leukemia. In Leukemia 13(3):393–9 (1999), Tamaki et al. reported that the Wilms' tumor gene WT1 is a good marker for diagnosis of disease progression of myelodysplastic syndromes. Expression of the Wilms' tumor gene WT1 in solid tumors, and its involvement in tumor cell growth, was discussed in relation to gastric cancer, colon cancer, lung cancer, breast cancer cell lines, germ cell tumor cell line, ovarian cancer, the uterine cancer, thyroid cancer cell line, hepatocellular carcinoma, in Oji et al., Jpn. J. Cancer Res. 90(2):194–204 (1999).

The compositions provided herein may be used alone or in combination with conventional therapeutic regimens such as surgery, irradiation, chemotherapy and/or bone marrow transplantation (autologous, syngeneic, allogeneic or unrelated). As discussed in greater detail below, binding agents and T cells as provided herein may be used for purging of autologous stem cells. Such purging may be beneficial prior to, for example, bone marrow transplantation or transfusion of blood or components thereof. Binding agents, T cells, antigen presenting cells (APC) and compositions provided herein may further be used for expanding and stimulating (or priming) autologous, allogeneic, syngeneic or unrelated WT1-specific T-cells in vitro and/or in vivo. Such WT1-specific T cells may be used, for example, within donor lymphocyte infusions.

Routes and frequency of administration, as well as dosage, will vary from individual to individual, and may be readily established using standard techniques. In general, the pharmaceutical compositions and vaccines may be administered by injection (e.g., intracutaneous, intramuscular, intravenous or subcutaneous), intranasally (e.g., by aspiration) or orally. In some tumors, pharmaceutical compositions or vaccines may be administered locally (by, for example, rectocoloscopy, gastroscopy, videoendoscopy, angiography or other methods known in the art). Preferably, between 1 and 10 doses may be administered over a 52 week period. Preferably, 6 doses are administered, at intervals of 1 month, and booster vaccinations may be given periodically thereafter. Alternate protocols may be appropriate for individual patients. A suitable dose is an amount of a compound that, when administered as described above, is capable of promoting an anti-tumor immune response that is at least 10–50% above the basal (i.e., untreated) level. Such response can be monitored by measuring the anti-tumor antibodies in a patient or by vaccine-dependent generation of cytolytic effector cells capable of killing the patient's tumor cells in vitro. Such vaccines should also be capable of causing an immune response that leads to an improved clinical outcome (e.g., more frequent complete or partial remissions, or longer disease-free and/or overall survival) in vaccinated patients as compared to non-vaccinated patients. In general, for pharmaceutical compositions and vaccines comprising one or more polypeptides, the amount of each polypeptide present in a dose ranges from about 100 μg to 5 mg. Suitable dose sizes will vary with the size of the patient, but will typically range from about 0.1 mL to about 5 mL.

In general, an appropriate dosage and treatment regimen provides the active compound(s) in an amount sufficient to provide therapeutic and/or prophylactic benefit. Such a response can be monitored by establishing an improved clinical outcome (e.g., more frequent complete or partial remissions, or longer disease-free and/or overall survival) in treated patients as compared to non-treated patients. Increases in preexisting immune responses to WT1 generally correlate with an improved clinical outcome. Such immune responses may generally be evaluated using standard proliferation, cytotoxicity or cytokine assays, which may be performed using samples obtained from a patient before and after treatment.

Within further aspects, methods for inhibiting the development of a malignant disease associated with WT1 expression involve the administration of autologous T cells that have been activated in response to a WT1 polypeptide or WT1-expressing APC, as described above. Such T cells may be CD4⁺ and/or CD8⁺, and may be proliferated as described above. The T cells may be administered to the individual in an amount effective to inhibit the development of a malignant disease. Typically, about 1×10⁹ to 1×10¹¹ T cells/M² are administered intravenously, intracavitary or in the bed of a resected tumor. It will be evident to those skilled in the art that the number of cells and the frequency of administration will be dependent upon the response of the patient.

Within certain embodiments, T cells may be stimulated prior to an autologous bone marrow transplantation. Such stimulation may take place in vivo or in vitro. For in vitro stimulation, bone marrow and/or peripheral blood (or a fraction of bone marrow or peripheral blood) obtained from a patient may be contacted with a WT1 polypeptide, a polynucleotide encoding a WT1 polypeptide and/or an APC that expresses a WT1 polypeptide under conditions and for a time sufficient to permit the stimulation of T cells as described above. Bone marrow, peripheral blood stem cells and/or WT1-specific T cells may then be administered to a patient using standard techniques.

Within related embodiments, T cells of a related or unrelated donor may be stimulated prior to a syngeneic or allogeneic (related or unrelated) bone marrow transplantation. Such stimulation may take place in vivo or in vitro. For in vitro stimulation, bone marrow and/or peripheral blood (or a fraction of bone marrow or peripheral blood) obtained from a related or unrelated donor may be contacted with a WT1 polypeptide, WT1 polynucleotide and/or APC that expresses a WT1 polypeptide under conditions and for a time sufficient to permit the stimulation of T cells as described above. Bone marrow, peripheral blood stem cells and/or WT1-specific T cells may then be administered to a patient using standard techniques.

Within other embodiments, WT1-specific T cells as described herein may be used to remove cells expressing WT1 from autologous bone marrow, peripheral blood or a fraction of bone marrow or peripheral blood (e.g., CD34⁺ enriched peripheral blood (PB) prior to administration to a patient). Such methods may be performed by contacting bone marrow or PB with such T cells under conditions and for a time sufficient to permit the reduction of WT1 expressing cells to less than 10%, preferably less than 5% and more preferably less than 1%, of the total number of myeloid or lymphatic cells in the bone marrow or peripheral blood. The extent to which such cells have been removed may be readily determined by standard methods such as, for example, qualitative and quantitative PCR analysis, morphology, immunohistochemistry and FACS analysis. Bone marrow or PB (or a fraction thereof) may then be administered to a patient using standard techniques.

Diagnostic Methods

The present invention further provides methods for detecting a malignant disease associated with WT1 expression, and for monitoring the effectiveness of an immunization or therapy for such a disease. Such methods are based on the discovery, within the present invention, that an immune response specific for WT1 protein can be detected in patients afflicted with such diseases, and that methods which enhance such immune responses may provide a preventive or therapeutic benefit.

To determine the presence or absence of a malignant disease associated with WT1 expression, a patient may be tested for the level of T cells specific for WT1. Within certain methods, a biological sample comprising CD4⁺ and/or CD8⁺ T cells isolated from a patient is incubated with a WT1 polypeptide, a polynucleotide encoding a WT1 polypeptide and/or an APC that expresses a WT1 polypeptide, and the presence or absence of specific activation of the T cells is detected, as described herein. Suitable biological samples include, but are not limited to, isolated T cells. For example, T cells may be isolated from a patient by routine techniques (such as by Ficoll/Hypaque density gradient centrifugation of peripheral blood lymphocytes). T cells may be incubated in vitro for 2–9 days (typically 4 days) at 37° C. with WT1 polypeptide (e.g., 5–25 μg/ml). It may be desirable to incubate another aliquot of a T cell sample in the absence of WT1 polypeptide to serve as a control. For CD4⁺ T cells, activation is preferably detected by evaluating proliferation of the T cells. For CD8⁺ T cells, activation is preferably detected by evaluating cytolytic activity. A level of proliferation that is at least two fold greater and/or a level of cytolytic activity that is at least 20% greater than in disease-free patients indicates the presence of a malignant disease associated with WT1 expression. Further correlation may be made, using methods well known in the art, between the level of proliferation and/or cytolytic activity and the predicted response to therapy. In particular, patients that display a higher antibody, proliferative and/or lytic response may be expected to show a greater response to therapy.

Within other methods, a biological sample obtained from a patient is tested for the level of antibody specific for WT1. The biological sample is incubated with a WT1 polypeptide, a polynucleotide encoding a WT1 polypeptide and/or an APC that expresses a WT1 polypeptide under conditions and for a time sufficient to allow immunocomplexes to form. Immunocomplexes formed between the WT1 polypeptide and antibodies in the biological sample that specifically bind to the WT1 polypeptide are then detected. A biological sample for use within such methods may be any sample obtained from a patient that would be expected to contain antibodies. Suitable biological samples include blood, sera, ascites, bone marrow, pleural effusion, and cerebrospinal fluid.

The biological sample is incubated with the WT1 polypeptide in a reaction mixture under conditions and for a time sufficient to permit immunocomplexes to form between the polypeptide and antibodies specific for WT1. For example, a biological sample and WT1 polypeptide may be incubated at 4° C. for 24–48 hours.

Following the incubation, the reaction mixture is tested for the presence of immunocomplexes. Detection of immunocomplexes formed between the WT1 polypeptide and antibodies present in the biological sample may be accomplished by a variety of known techniques, such as radioimmunoassays (RIA) and enzyme linked immunosorbent assays (ELISA). Suitable assays are well known in the art and are amply described in the scientific and Patent literature (e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988). Assays that may be used include, but are not limited to, the double monoclonal antibody sandwich immunoassay technique of David et al. (U.S. Pat. No. 4,376,110); monoclonal-polyclonal antibody sandwich assays (Wide et al., in Kirkham and Hunter, eds., Radioimmunoassay Methods, E. and S. Livingstone, Edinburgh, 1970); the “western blot” method of Gordon et al. (U.S. Pat. No. 4,452,901); immunoprecipitation of labeled ligand (Brown et al., J. Biol. Chem. 255:4980–4983, 1980); enzyme-linked immunosorbent assays as described by, for example, Raines and Ross (J. Biol. Chem. 257:5154–5160, 1982); immunocytochemical techniques, including the use of fluorochromes (Brooks et al., Clin. Exp. Immunol. 39: 477, 1980); and neutralization of activity (Bowen-Pope et al., Proc. Natl. Acad. Sci. USA 81:2396–2400, 1984). Other immunoassays include, but are not limited to, those described in U.S. Pat. Nos.: 3,817,827; 3,850,752; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; and 4,098,876.

For detection purposes, WT1 polypeptide may either be labeled or unlabeled. Unlabeled WT1 polypeptide may be used in agglutination assays or in combination with labeled detection reagents that bind to the immunocomplexes (e.g., anti-immunoglobulin, protein G, protein A or a lectin and secondary antibodies, or antigen-binding fragments thereof, capable of binding to the antibodies that specifically bind to the WT1 polypeptide). If the WT1 polypeptide is labeled, the reporter group may be any suitable reporter group known in the art, including radioisotopes, fluorescent groups, luminescent groups, enzymes, biotin and dye particles.

Within certain assays, unlabeled WT1 polypeptide is immobilized on a solid support. The solid support may be any material known to those of ordinary skill in the art to which the polypeptide may be attached. For example, the solid support may be a test well in a microtiter plate or a nitrocellulose or other suitable membrane. Alternatively, the support may be a bead or disc, such as glass, fiberglass, latex or a plastic material such as polystyrene or polyvinylchloride. The support may also be a magnetic particle or a fiber optic sensor, such as those disclosed, for example, in U.S. Pat. No. 5,359,681. The polypeptide may be immobilized on the solid support using a variety of techniques known to those of skill in the art, which are amply described in the Patent and scientific literature. In the context of the present invention, the term “immobilization” refers to both noncovalent association, such as adsorption, and covalent attachment (which may be a direct linkage between the antigen and functional groups on the support or may be a linkage by way of a cross-linking agent). Immobilization by adsorption to a well in a microtiter plate or to a membrane is preferred. In such cases, adsorption may be achieved by contacting the WT1 polypeptide, in a suitable buffer, with the solid support for a suitable amount of time. The contact time varies with temperature, but is typically between about 1 hour and about 1 day. In general, contacting a well of a plastic microtiter plate (such as polystyrene or polyvinylchloride) with an amount of polypeptide ranging from about 10 ng to about 10 μg, and preferably about 100 ng to about 1 μg, is sufficient to immobilize an adequate amount of polypeptide.

Following immobilization, the remaining protein binding sites on the support are typically blocked. Any suitable blocking agent known to those of ordinary skill in the art, such as bovine serum albumin, Tween 20™ (Sigma Chemical Co., St. Louis, Mo.), heat-inactivated normal goat serum (NGS), or BLOTTO (buffered solution of nonfat dry milk which also contains a preservative, salts, and an antifoaming agent). The support is then incubated with a biological sample suspected of containing specific antibody. The sample can be applied neat, or, more often, it can be diluted, usually in a buffered solution which contains a small amount (0.1%–5.0% by weight) of protein, such as BSA, NGS, or BLOTTO. In general, an appropriate contact time (i.e., incubation time) is a period of time that is sufficient to detect the presence of antibody that specifically binds WT1 within a sample containing such an antibody. Preferably, the contact time is sufficient to achieve a level of binding that is at least about 95% of that achieved at equilibrium between bound and unbound antibody. Those of ordinary skill in the art will recognize that the time necessary to achieve equilibrium may be readily determined by assaying the level of binding that occurs over a period of time. At room temperature, an incubation time of about 30 minutes is generally sufficient.

Unbound sample may then be removed by washing the solid support with an appropriate buffer, such as PBS containing 0.1% Tween 20™. A detection reagent that binds to the immunocomplexes and that comprises a reporter group may then be added. The detection reagent is incubated with the immunocomplex for an amount of time sufficient to detect the bound antibody. An appropriate amount of time may generally be determined by assaying the level of binding that occurs over a period of time. Unbound detection reagent is then removed and bound detection reagent is detected using the reporter group. The method employed for detecting the reporter group depends upon the nature of the reporter group. For radioactive groups, scintillation counting or autoradiographic methods are generally appropriate. Spectroscopic methods may be used to detect dyes, luminescent groups and fluorescent groups. Biotin may be detected using avidin, coupled to a different reporter group (commonly a radioactive or fluorescent group or an enzyme). Enzyme reporter groups (e.g., horseradish peroxidase, beta-galactosidase, alkaline phosphatase and glucose oxidase) may generally be detected by the addition of substrate (generally for a specific period of time), followed by spectroscopic or other analysis of the reaction products. Regardless of the specific method employed, a level of bound detection reagent that is at least two fold greater than background (i.e., the level observed for a biological sample obtained from a disease-free individual) indicates the presence of a malignant disease associated with WT1 expression.

In general, methods for monitoring the effectiveness of an immunization or therapy involve monitoring changes in the level of antibodies or T cells specific for WT1 in the patient. Methods in which antibody levels are monitored may comprise the steps of: (a) incubating a first biological sample, obtained from a patient prior to a therapy or immunization, with a WT1 polypeptide, wherein the incubation is performed under conditions and for a time sufficient to allow immunocomplexes to form; (b) detecting immunocomplexes formed between the WT1 polypeptide and antibodies in the biological sample that specifically bind to the WT1 polypeptide; (c) repeating steps (a) and (b) using a second biological sample taken from the patient following therapy or immunization; and (d) comparing the number of immunocomplexes detected in the first and second biological samples. Alternatively, a polynucleotide encoding a WT1 polypeptide, or an APC expressing a WT1 polypeptide may be employed in place of the WT1 polypeptide. Within such methods, immunocomplexes between the WT1 polypeptide encoded by the polynucleotide, or expressed by the APC, and antibodies in the biological sample are detected.

Methods in which T cell activation and/or the number of WT1 specific precursors are monitored may comprise the steps of: (a) incubating a first biological sample comprising CD4+ and/or CD8+ cells (e.g., bone marrow, peripheral blood or a fraction thereof), obtained from a patient prior to a therapy or immunization, with a WT1 polypeptide, wherein the incubation is performed under conditions and for a time sufficient to allow specific activation, proliferation and/or lysis of T cells; (b) detecting an amount of activation, proliferation and/or lysis of the T cells; (c) repeating steps (a) and (b) using a second biological sample comprising CD4+ and/or CD8+ T cells, and taken from the same patient following therapy or immunization; and (d) comparing the amount of activation, proliferation and/or lysis of T cells in the first and second biological samples. Alternatively, a polynucleotide encoding a WT1 polypeptide, or an APC expressing a WT1 polypeptide may be employed in place of the WT1 polypeptide.

A biological sample for use within such methods may be any sample obtained from a patient that would be expected to contain antibodies, CD4+ T cells and/or CD8+ T cells. Suitable biological samples include blood, sera, ascites, bone marrow, pleural effusion and cerebrospinal fluid. A first biological sample may be obtained prior to initiation of therapy or immunization or part way through a therapy or vaccination regime. The second biological sample should be obtained in a similar manner, but at a time following additional therapy or immunization. The second biological sample may be obtained at the completion of, or part way through, therapy or immunization, provided that at least a portion of therapy or immunization takes place between the isolation of the first and second biological samples.

Incubation and detection steps for both samples may generally be performed as described above. A statistically significant increase in the number of immunocomplexes in the second sample relative to the first sample reflects successful therapy or immunization.

The following Examples are offered by way of illustration and not by way of limitation.

EXAMPLES Example 1 Identification of an Immune Response to WT1 in Patients with Hematological Malignancies

This Example illustrates the identification of an existent immune response in patients with a hematological malignancy.

To evaluate the presence of preexisting WT1 specific antibody responses in patients, sera of patients with acute myelogenous leukemia (AML), acute lymphocytic leukemia (ALL), chronic myelogenous leukemia (CML) and severe aplastic anemia were analyzed using Western blot analysis. Sera were tested for the ability to immunoprecipitate WT1 from the human leukemic cell line K562 (American Type Culture Collection, Manassas, Va.). In each case, immunoprecipitates were separated by gel electrophoresis, transferred to membrane and probed with the anti WT-1 antibody WT180 (Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.). This Western blot analysis identified potential WT1 specific antibodies in patients with hematological malignancy. A representative Western blot showing the results for a patient with AML is shown in FIG. 2. A 52 kD protein in the immunoprecipitate generated using the patient sera was recognized by the WT1 specific antibody. The 52 kD protein migrated at the same size as the positive control.

Additional studies analyzed the sera of patients with AML and CML for the presence of antibodies to full-length and truncated WT1 proteins. CDNA constructs representing the human WT1/full-length (aa 1–449), the N-terminus (aa 1–249) (WT1/N-terminus) and C-terminuis (aa 267–449) (WT1/C-terminus) region were subcloned into modified pET28 vectors. The WT1/full-length and WT1/N-terminus proteins were expressed as Ra12 fusion proteins. Ra12 is the C-terminal fragment of a secreted Mycobacterium tuberculosis protein, denoted as MTB32B. (Skeiky et al., Infect Immun. 67;3998, 1999). The Ra12-WT1/full-length fusion region was cloned 3′ to a histidine-tag in a histidine-tag modified pET28 vector. The WT1/N-terminus region was subcloned into a modified pET28 vector that has a 5′ histidine-tag followed by the thioredoxin (TRX)-WT1/N-terminus fusion region followed by a 3′ histidine-tag. The WT1/C-terminus coding region was subcloned into a modified pET28 vector without a fusion partner containing only the 5′ and 3′ histidine-tag, followed by a Thrombin and EK site.

BL21 pLysS E. coli (Stratagene, La Jolla, Calif.) were transformed with the three WT1 expression constructs, grown overnight and induced with isopropyl-β-D-thiogalactoside (IPTG). WT1 proteins were purified as follows: Cells were harvested and lysed by incubation in 10 mM Tris, pH 8.0 with Complete Protease Inhibitor Tablets (Boehringer Mannheim Biochemicals, Indianapolis, Ind.) at 37° C. followed by repeated rounds of sonication. Inclusion bodies were washed twice with 10 mM Tris, pH 8.0. Proteins were then purified by metal chelate affinity chromatography over nickel-nitrilotriacetic acid resin (QIAGEN Inc., Valencia, Calif.; Hochuli et al., Biologically Active Molecules: 217, 1989) followed by chromatography on a Source Q anion exchange resin (Amersham Pharmacia Biotech, Upsala, Sweden). The identity of the WT1 proteins was confirmed by N-terminal sequencing.

Sera from adult patients with de nova AML or CML were studied for the presence of WT1 specific Ab. Recombinant proteins were adsorbed to TC microwell plates (Nunc, Roskilde, Denmark). Plates were washed with PBS/0.5% Tween 20 and blocked with 1% BSA/PBS/0.1% Tween 20. After washing, serum dilutions were added and incubated overnight at 4° C. Plates were washed and Donkey anti-human IgG-HRP secondary antibody was added (Jackson-Immunochem, West Grove, Pa.) and incubated for 2h at room temperature. Plates were washed, incubated with TMB Peroxidase substrate solution (Kirkegaard and Perry Laboratories, Mass.), quenched with 1N H₂SO₄, and immediately read (Cyto-Fluor 2350; Millipore, Bedford, Mass.).

For the serological survey, human sera were tested by ELISA over a range of serial dilutions from 1:50 to 1:20,000. A positive reaction was defined as an OD value of a 1:500 diluted serum that exceeded the mean OD value of sera from normal donors (n=96) by three (WT1/full-length, WT1C-terminus) standard deviations. Due to a higher background in normal donors to the WT1/N-terminus protein a positive reaction to WT1/N-terninus was defined as an OD value of 1:500 diluted serum that exceeded the mean OD value of sera from normal donors by four standard deviations. To verify that the patient Ab response was directed against WT1 and not to the Ra12 or TRX fusion part of the protein or possible E. coli contaminant proteins, controls included the Ra12 and TRX protein alone purified in a similar manner. Samples that showed reactivity against the Ra12 and/or TRX proteins were excluded from the analysis.

To evaluate for the presence of immunity to WT1, Ab to recombinant full-length and truncated WT1 proteins in the sera of normal individuals and patients with leukemia were determined. Antibody reactivity was analyzed by ELISA reactivity to WT1/full-length protein, WT1/N-terminus protein and WT1/C-terminus protein.

Only 2 of 96 normal donors had serum antibodies reactive with WT1/full-length protein (FIG. 18). One of those individuals had antibody to WT1/N-terminus protein and one had antibody to WT1/C-terminus protein. In contrast, 16 of 63 patients (25%) with AML had serum antibodies reactive with WT1/full-length protein. By marked contrast, only 2 of 63 patients (3%) had reactivity to WT1/C-terminus protein. Fifteen of 81 patients (19%) with CML had serum antibodies reactive with WT1/full-length protein and 12 of 81 patients (15%) had serum antibodies reactive with WT1/N-terminus. Only 3 of 81 patients (3%) had reactivity to WT1/C-terminus protein. (FIGS. 16 and 17.)

These data demonstrate that Ab responses to WT1 are detectable in some patients with AML and CML. The greater incidence of antibody in leukemia patients provides strong evidence that immunization to the WT1 protein occurred as a result of patients bearing malignancy that expresses or at some time expressed WT1. Without being limited to a specific theory, it is believed that the observed antibody responses to WT1 most probably result from patients becoming immune to WT1 on their own leukemia cells and provide direct evidence that WT1 can be immunogenic despite being a “self” protein.

The presence of antibody to WT1 strongly implies that concurrent helper T cell responses are also present in the same patients. WT1 is an internal protein. Thus, CTL responses are likely to be the most effective in terms of leukemia therapy and the most toxic arm of immunity. Thus, these data provide evidence that therapeutic vaccines directed against WT1 will be able to elicit an immune response to WT1.

The majority of the antibodies detected were reactive with epitopes within the N-terminus while only a small subgroup of patients showed a weak antibody response to the C-terminus. This is consistent with observations in the animal model, where immunization with peptides derived from the N-terminus elicited antibody, helper T cell and CTL responses, whereas none of the peptides tested from the C-terminus elicited antibody or T cell responses (Gaiger et al., Blood 96:1334, 2000).

Example 2 Induction of Antibodies to WT1 in Mice Immunized with Cell Lines Expressing WT1

This Example illustrates the use of cells expressing WT1 to induce a WT1 specific antibody response in vivo.

Detection of existent antibodies to WT1 in patients with leukemia strongly implied that it is possible to immunize to WT1 protein to elicit immunity to WT1. To test whether immunity to WT1 can be generated by vaccination, mice were injected with TRAMP-C, a WT1 positive tumor cell line of B6 origin. Briefly, male B6 mice were immunized with 5×10 ⁶ TRAMP-C cells subcutaneously and boosted twice with 5×10⁶ cells at three week intervals. Three weeks after the final immunization, sera were obtained and single cell suspensions of spleens were prepared in RPMI 1640 medium (GIBCO) with 25 μM β-2-mercaptoethanol, 200 units of penicillin per ml, 10 mM L-glutamine, and 10% fetal bovine serum.

Following immunization to TRAMP-C, a WT1 specific antibody response in the immunized animals was detectable. A representative Western blot is shown in FIG. 3. These results show that immunization to WT1 protein can elicit an immune response to WT1 protein.

Example 3

Induction of Th and Antibody Responses in Mice Immunized with WT1 Peptides

This Example illustrates the ability of immunization with WT1 peptides to elicit an immune response specific for WT1.

Peptides suitable for eliciting Ab and proliferative T cell responses were identified according to the Tsites program (Rothbard and Taylor, EMBO J. 7:93–100, 1988; Deavin et al., Mol. Immunol. 33:145–155, 1996), which searches for peptide motifs that have the potential to elicit Th responses. Peptides shown in Table I were synthesized and sequenced.

TABLE I WT1 Peptides Peptide Sequence Comments Mouse: p6–22 RDLNALLPAVSSLGGGG 1 mismatch relative to (SEQ ID NO:13) human WT1 sequence Human: p6–22 RDLNALLPAVPSLGGGG (SEQ ID NO:1) Human/mouse: PSQASSGQARMFPNAPYLPSCLE p117–139 (SEQ ID NOs: 2 and 3) Mouse: p244–262 GATLKGMAAGSSSSVKWTE 1 mismatch relative to (SEQ ID NO:14) human WT1 sequence Human: p244–262 GATLKGVAAGSSSSVKWTE (SEQ ID NO:4) Human/mouse: RIHTHGVFRGIQDVR p287–301 (SEQ ID NOs: 15 and 16) Mouse: p299–313 VRRVSGVAPTLVRS 1 mismatch relative to (SEQ ID NO:17) human WT1 sequence Human/mouse: CQKKFARSDELVRHH p421–435 (SEQ ID NOs: 19 and 20) For immunization, peptides were grouped as follows: Group A: p6–22 human: 10.9 mg in 1 ml (10 μl = 100 μg)          p117–139 human/mouse: 7.6 mg in 1 ml (14 μl = 100 μg)          p244–262 human: 4.6. mg in 1 ml (22 μl = 100 μg) Group B: p287–301 human/mouse: 7.2 mg in 1 ml (14 μl = 100 μg)          mouse p299–313: 6.6. mg in 1 ml (15 μl = 100 μg)          p421–435 human/mouse: 3.3 mg in 1 ml (30 μl = 100 μg) Control: (FBL peptide 100 μg) + CFA/IFA Control: (CD45 peptide 100 μg) + CFA/IFA

Group A contained peptides present within the amino terminus portion of WT1 (exon 1) and Group B contained peptides present within the carboxy terminus, which contains a four zinc finger region with sequence homology to other DNA-binding proteins. Within group B, p287–301 and p299–313 were derived from exon 7, zinc finger 1, and p421–435 was derived from exon 10, zinc finger IV.

B6 mice were immunized with a group of WT1 peptides or with a control peptide. Peptides were dissolved in 1 ml sterile water for injection, and B6 mice were immunized 3 times at time intervals of three weeks. Adjuvants used were CFA/IFA, GM-CSF, and Montinide. The presence of antibodies specific for WT1 was then determined as described in Examples 1 and 2, and proliferative T cell responses were evaluated using a standard thymidine incorporation assay, in which cells were cultured in the presence of antigen and proliferation was evaluated by measuring incorporated radioactivity (Chen et al., Cancer Res. 54:1065–1070, 1994). In particular, lymphocytes were cultured in 96-well plates at 2×10⁵ cells per well with 4×10⁵ irradiated (3000 rads) syngeneic spleen cells and the designated peptide.

Immunization of mice with the group of peptides designated as Group A elicited an antibody response to WT1 (FIG. 4). No antibodies were detected following immunization to Vaccine B, which is consistent with a lack of helper T cell response from immunization with Vaccine B. P117–139 elicited proliferative T cell responses (FIGS. 5A–5C). The stimulation indices (SI) varied between 8 and 72. Other peptides (P6–22 and P299–313) also were shown to elicit proliferative T cell responses. Immunization with P6–22 resulted in a stimulation index (SI) of 2.3 and immunization with P299–313 resulted in a SI of 3.3. Positive controls included ConA stimulated T cells, as well as T cells stimulated with known antigens, such as CD45 and FBL, and allogeneic T cell lines (DeBruijn et al., Eur. J. Immunol. 21:2963–2970, 1991).

FIGS. 6A and 6B show the proliferative response observed for each of the three peptides within vaccine A (FIG. 6A) and vaccine B (FIG. 6B). Vaccine A elicited proliferative T cell responses to the immunizing peptides p6-22 and p117–139, with stimulation indices (SI) varying between 3 and 8 (bulk lines). No proliferative response to p244–262 was detected (FIG. 6A).

Subsequent in vitro stimulations were carried out as single peptide stimulations using only p6-22 and p117–139. Stimulation of the Vaccine A specific T cell line with p117–139 resulted in proliferation to p117–139 with no response to p6-22 (FIG. 7A). Clones derived from the line were specific for p117–139 (FIG. 7B). By contrast, stimulation of the Vaccine A specific T cell line with p6–22 resulted in proliferation to p6-22 with no response to p117–139 (FIG. 7C). Clones derived from the line were specific for p6-22 (FIG. 7D).

These results show that vaccination with WT1 peptides can elicit antibody responses to WT1 protein and proliferative T cell responses to the immunizing peptides.

Example 4 Induction of CTL Responses in Mice Immunized with WT1 Peptides

This Example illustrates the ability of WT1 peptides to elicit CTL immunity.

Peptides (9-mers) with motifs appropriate for binding to class I MHC were identified using a BIMAS HLA peptide binding prediction analysis (Parker et al., J. Immunol. 152:163, 1994). Peptides identified within such analyses are shown in Tables II–XLIV. In each of these tables, the score reflects the theoretical binding affinity (half-time of dissociation) of the peptide to the MHC molecule indicated.

Peptides identified using the Tsites program (Rothbard and Taylor, EMBO J. 7:93–100, 1988; Deavin et al., Mol. Immunol. 33:145–155, 1996), which searches for peptide motifs that have the potential to elicit Th responses are further shown in FIGS. 8A and 8B, and Table XLV.

TABLE II Results of BIMAS HLA Peptide Binding Prediction Analysis for Binding of Human WT1 Peptides to Human HLA A1 Score (Estimate of Half Time of Disassociation of a Start Molecule Containing This Rank Position Subsequence Residue Listing Subsequence) 1 137 CLESQPAIR (SEQ ID NO:47)  18.000 2 80 GAEPHEEQC (SEQ ID NO:87)  9.000 3 40 FAPPGASAY (SEQ ID NO:74)  5.000 4 354 QCDFKDCER (SEQ ID NO:162) 5.000 5 2 GSDVRDLNA (SEQ ID NO:101) 3.750 6 152 VTFDGTPSY (SEQ ID NO:244) 2.500 7 260 WTEGQSNHS (SEQ ID NO:247) 2.250 8 409 TSEKPFSCR (SEQ ID NO:232) 1.350 9 73 KQEPSWGGA (SEQ ID NO:125) 1.350 10 386 KTCQRKFSR (SEQ ID NO:128) 1.250 11 37 VLDFAPPGA (SEQ ID NO:241) 1.000 12 325 CAYPGCNKR (SEQ ID NO:44)  1.000 13 232 QLECMTWNQ (SEQ ID NO:167) 0.900 14 272 ESDNHTTPI (SEQ ID NO:71)  0.750 15 366 RSDQLKRHQ (SEQ ID NO:193) 0.750 16 222 SSDNLYQMT (SEQ ID NO:217) 0.750 17 427 RSDELVRHH (SEQ ID NO:191) 0.750 18 394 RSDHLKTHT (SEQ ID NO:192) 0.750 19 317 TSEKRPFMC (SEQ ID NO:233) 0.675 20 213 QALLLRTPY (SEQ ID NO:160) 0.500

TABLE III Results of BIMAS HLA Peptide Binding Prediction Analysis for Binding of Human WT1 Peptides to Human HLA A 0201 Score (Estimate of Half Time of Disassociation of a Start Molecule Containing This Rank Position Subsequence Residue Listing Subsequence) 1 126 RMFPNAPYL (SEQ ID NO:185) 313.968 2 187 SLGEQQYSV (SEQ ID NO:214) 285.163 3 10 ALLPAVPSL (SEQ ID NO:34)  181.794 4 242 NLGATLKGV (SEQ ID NO:146) 159.970 5 225 NLYQMTSQL (SEQ ID NO:147) 68.360 6 292 GVFRGIQDV (SEQ ID NO:103) 51.790 7 191 QQYSVPPPV (SEQ ID NO:171) 22.566 8 280 ILCGAQYRI (SEQ ID NO:116) 17.736 9 235 CMTWNQMNL (SEQ ID NO:49)  15.428 10 441 NMTKLQLAL (SEQ ID NO:149) 15.428 11 7 DLNALLPAV (SEQ ID NO:58)  11.998 12 227 YQMTSQLEC (SEQ ID NO:251) 8.573 13 239 NQMNLGATL (SEQ ID NO:151) 8.014 14 309 TLVRSASET (SEQ ID NO:226) 7.452 15 408 KTSEKPFSC (SEQ ID NO:129) 5.743 16 340 LQMHSRKHT (SEQ ID NO:139) 4.752 17 228 QMTSQLECM (SEQ ID NO:169) 4.044 18 93 TVHFSGQFT (SEQ ID NO:235) 3.586 19 37 VLDFAPPGA (SEQ ID NO:241) 3.378 20 86 EQCLSAFTV (SEQ ID NO:69)  3.068

TABLE IV Results of BIMAS HLA Peptide Binding Prediction Analysis for Binding of Human WT1 Peptides to Human HLA A 0205 Score (Estimate of Half Time of Disassociation of a Start Molecule Containing This Rank Position Subsequence Residue Listing Subsequence) 1 10 ALLPAVPSL (SEQ ID NO:34)  42.000 2 292 GVFRGIQDV (SEQ ID NO:103) 24.000 3 126 RMFPNAPYL (SEQ ID NO:185) 21.000 4 225 NLYQMTSQL (SEQ ID NO:147) 21.000 5 239 NQMNLGATL (SEQ ID NO:151) 16.800 6 302 RVPGVAPTL (SEQ ID NO:195) 14.000 7 441 NMTKLQLAL (SEQ ID NO:149) 7.000 8 235 CMTWNQMNL (SEQ ID NO:49)  7.000 9 187 SLGEQQYSV (SEQ ID NO:214) 6.000 10 191 QQYSVPPPV (SEQ ID NO:171) 4.800 11 340 LQMHSRKHT (SEQ ID NO:139) 4.080 12 242 NLGATLKGV (SEQ ID NO:146) 4.000 13 227 YQMTSQLEC (SEQ ID NO:251) 3.600 14 194 SVPPPVYGC (SEQ ID NO:218) 2.000 15 93 TVHFSGQFT (SEQ ID NO:235) 2.000 16 280 ILCGAQYRI (SEQ ID NO:116) 1.700 17 98 GQFTGTAGA (SEQ ID NO:99)  1.200 18 309 TLVRSASET (SEQ ID NO:226) 1.000 19 81 AEPHEEQCL (SEQ ID NO:30)  0.980 20 73 KQEPSWGGA (SEQ ID NO:125) 0.960

TABLE V Results of BIMAS HLA Peptide Binding Prediction Analysis for Binding of Human WT1 Peptides to Human HLA A24 Score (Estimate of Half Time of Disassociation of a Start Molecule Containing This Rank Position Subsequence Residue Listing Subsequence) 1 302 RVPGVAPTL (SEQ ID NO:195) 16.800 2 218 RTPYSSDNL (SEQ ID NO:194) 12.000 3 356 DFKDCERRF (SEQ ID NO:55)  12.000 4 126 RMFPNAPYL (SEQ ID NO:185) 9.600 5 326 AYPGCNKRY (SEQ ID NO:42)  7.500 6 270 GYESDNHT (SEQ ID NO:106)T 7.500 7 239 NQMNLGATL (SEQ ID NO:151) 7.200 8 10 ALLPAVPSL (SEQ ID NO:34)  7.200 9 130 NAPYLPSCL (SEQ ID NO:144) 7.200 10 329 GCNKRYFKL (SEQ ID NO:90)  6.600 11 417 RWPSCQKKF (SEQ ID NO:196) 6.600 12 47 AYGSLGGPA (SEQ ID NO:41)  6.000 13 180 DPMGQQGSL (SEQ ID NO:59)  6.000 14 4 DVRDLNALL (SEQ ID NO:62)  5.760 15 285 QYRIHTHGV (SEQ ID NO:175) 5.000 16 192 QYSVPPPVY (SEQ ID NO:176) 5.000 17 207 DSCTGSQAL (SEQ ID NO:61)  4.800 18 441 NMTKLQLAL (SEQ ID NO:149) 4.800 19 225 NLYQMTSQL (SEQ ID NO:147) 4.000 20 235 CMTWNQMNL (SEQ ID NO:49)  4.000

TABLE VI Results of BIMAS HLA Peptide Binding Prediction Analysis for Binding of Human WT1 Peptides to Human HLA A3 Score (Estimate of Half Time of Disassociation of a Start Molecule Containing This Rank Position Subsequence Residue Listing Subsequence) 1 436 NMHQRNMTK (SEQ ID NO:148) 40.000 2 240 QMNLGATLK (SEQ ID NO:168) 20.000 3 88 CLSAFTVHF (SEQ ID NO:48)  6.000 4 126 RMFPNAPYL (SEQ ID NO:185) 4.500 5 169 AQFPNHSFK (SEQ ID NO:36)  4.500 6 10 ALLPAVPSL (SEQ ID NO:34)  4.050 7 137 CLESQPAIR (SEQ ID NO:47)  4.000 8 225 NLYQMTSQL (SEQ ID NO:147) 3.000 9 32 AQWAPVLDF (SEQ ID NO:37)  2.700 10 280 ILCGAQYRI (SEQ ID NO:116) 2.700 11 386 KTCQRKFSR (SEQ ID NO:128) 1.800 12 235 CMTWNQMNL (SEQ ID NO:49)  1.200 13 441 NMTKLQLAL (SEQ ID NO:149) 1.200 14 152 VTFDGTPSY (SEQ ID NO:244) 1.000 15 187 SLGEQQYSV (SEQ ID NO:214) 0.900 16 383 FQCKTCQRK (SEQ ID NO:80)  0.600 17 292 GVFRGIQDV (SEQ ID NO:103) 0.450 18 194 SVPPPVYGC (SEQ ID NO:218) 0.405 19 287 RIHTHGVFR (SEQ ID NO:182) 0.400 20 263 GQSNHSTGY (SEQ ID NO:100) 0.360

TABLE VII Results of BIIMAS HLA Peptide Binding Prediction Analysis for Binding of Human WT1 Peptides to Human HLA A68.1 Score (Estimate of Half Time of Disassociation of a Start Molecule Containing This Rank Position Subsequence Residue Listing Subsequence) 1 100 FTGTAGACR (SEQ ID NO:84)  100.000 2 386 KTCQRKFSR (SEQ ID NO:128) 50.000 3 368 DQLKRHQRR (SEQ ID NO:60)  30.000 4 312 RSASETSEK (SEQ ID NO:190) 18.000 5 337 LSHLQMHSR (SEQ ID NO:141) 15.000 6 364 FSRSDQLKR (SEQ ID NO:83)  15.000 7 409 TSEKPFSCR (SEQ ID NO:232) 15.000 8 299 DVRRVPGVA (SEQ ID NO:63)  12.000 9 4 DVRDLNALL (SEQ ID NO:62)  12.000 10 118 SQASSGQAR (SEQ ID NO:216) 10.000 11 343 HSRKHTGEK (SEQ ID NO:111) 9.000 12 169 AQFPNHSFK (SEQ ID NO:36)  9.000 13 292 GVFRGIQDV (SEQ ID NO:103) 8.000 14 325 CAYPGCNKR (SEQ ID NO:44)  7.500 15 425 FARSDELVR (SEQ ID NO:75)  7.500 16 354 QCDFKDCER (SEQ ID NO:162) 7.500 17 324 MCAYPGCNK (SEQ ID NO:142) 6.000 18 251 AAGSSSSVK (SEQ ID NO:28)  6.000 19 379 GVKPFQCKT (SEQ ID NO:104) 6.000 20 137 CLESQPAIR (SEQ ID NO:47)  5.000

TABLE VIII Results of BIMAS HLA Peptide Binding Prediction Analysis for Binding of Human WT1 Peptides to Human HLA A 1101 Score (Estimate of Half Time of Disassociation of a Start Molecule Containing This Rank Position Subsequence Residue Listing Subsequence) 1 386 KTCQRKFSR (SEQ ID NO:128) 1.800 2 169 AQFPNHSFK (SEQ ID NO:36)  1.200 3 436 NMHQRNMTK (SEQ ID NO:148) 0.800 4 391 KFSRSDHLK (SEQ ID NO:120) 0.600 5 373 HQRRHTGVK (SEQ ID NO:109) 0.600 6 383 FQCKTCQRK (SEQ ID NO:80)  0.600 7 363 RFSRSDQLK (SEQ ID NO:178) 0.600 8 240 QMNLGATLK (SEQ ID NO:168) 0.400 9 287 RIHTHGVFR (SEQ ID NO:182) 0.240 10 100 FTGTAGACR (SEQ ID NO:84)  0.200 11 324 MCAYPGCNK (SEQ ID NO:142) 0.200 12 251 AAGSSSSVK (SEQ ID NO:28)  0.200 13 415 SCRWPSCQK (SEQ ID NO:201) 0.200 14 118 SQASSGQAR (SEQ ID NO:216) 0.120 15 292 GVFRGIQDV (SEQ ID NO:103) 0.120 16 137 CLESQPAIR (SEQ ID NO:47)  0.080 17 425 FARSDELVR (SEQ ID NO:75)  0.080 18 325 CAYPGCNKR (SEQ ID NO:44)  0.080 19 312 RSASETSEK (SEQ ID NO:190) 0.060 20 65 PPPPHSFI (SEQ ID NO:156)K 0.060

TABLE IX Results of BIMAS HLA Peptide Binding Prediction Analysis for Binding of Human WT1 Peptides to Human HLA A 3101 Score (Estimate of Half Time of Disassociation of a Start Molecule Containing This Rank Position Subsequence Residue Listing Subsequence) 1 386 KTCQRKFSR (SEQ ID NO:128) 9.000 2 287 RIHTHGVFR (SEQ ID NO:182) 6.000 3 137 CLESQPAIR (SEQ ID NO:47)  2.000 4 118 SQASSGQAR (SEQ ID NO:216) 2.000 5 368 DQLKRHQRR (SEQ ID NO:60)  1.200 6 100 FTGTAGACR (SEQ ID NO:84)  1.000 7 293 VFRGIQDVR (SEQ ID NO:238) 0.600 8 325 CAYPGCNKR (SEQ ID NO:44)  0.600 9 169 AQFPNHSFK (SEQ ID NO:36)  0.600 10 279 PILCGAQYR (SEQ ID NO:155) 0.400 11 436 NMHQRNMTK (SEQ ID NO:148) 0.400 12 425 FARSDELVR (SEQ ID NO:75)  0.400 13 32 AQWAPVLDF (SEQ ID NO:37)  0.240 14 240 QMNLGATLK (SEQ ID NO:168) 0.200 15 354 QCDFKDCER (SEQ ID NO:162) 0.200 16 373 HQRRHTGVK (SEQ ID NO:109) 0.200 17 383 FQCKTCQRK (SEQ ID NO:80)  0.200 18 313 SASETSEKR (SEQ ID NO:197) 0.200 19 358 KDCERRFSR (SEQ ID NO:118) 0.180 20 391 KFSRSDHLK (SEQ ID NO:120) 0.180

TABLE X Results of BIMAS HLA Peptide Binding Prediction Analysis for Binding of Human WT1 Peptides to Human HLA A 3302 Score (Estimate of Half Time of Disassociation of a Start Molecule Containing This Rank Position Subsequence Residue Listing Subsequence) 1 337 LSHLQMHSR (SEQ ID NO:141) 15.000 2 409 TSEKPFSCR (SEQ ID NO:232) 15.000 3 364 FSRSDQLKR (SEQ ID NO:83) 15.000 4 137 CLESQPAIR (SEQ ID NO:47) 9.000 5 368 DQLKRHQRR (SEQ ID NO:60) 9.000 6 287 RIHTHGVFR (SEQ ID NO:182) 4.500 7 210 TGSQALLLR (SEQ ID NO:223) 3.000 8 425 FARSDELVR (SEQ ID NO:75) 3.000 9 313 SASETSEKR (SEQ ID NO:197) 3.000 10 293 VFRGIQDVR (SEQ ID NO:238) 3.000 11 354 QCDFKDCER (SEQ ID NO:162) 3.000 12 100 FTGTAGACR (SEQ ID NO:84) 3.000 13 118 SQASSGQAR (SEQ ID NO:216) 3.000 14 325 CAYPGCNKR (SEQ ID NO:44) 3.000 15 207 DSCTGSQAL (SEQ ID NO:61) 1.500 16 139 ESQPAIRNQ (SEQ ID NO:72) 1.500 17 299 DVRRVPGVA (SEQ ID NO:63) 1.500 18 419 PSCQKKFAR (SEQ ID NO:159) 1.500 19 272 ESDNHTTPI (SEQ ID NO:71) 1.500 20 4 DVRDLNALL (SEQ ID NO:62) 1.500

TABLE XI Results of BIMAS HLA Peptide Binding Prediction Analysis for Binding of Human WT1 Peptides to Human HLA B14 Score (Estimate of Half Time of Disassociation of a Start Molecule Containing This Rank Position Subsequence Residue Listing Subsequence) 1 362 RRFSRSDQL (SEQ ID NO:187) 1000.000 2 332 KRYFKLSHL (SEQ ID NO:127) 300.000 3 423 KKFARSDEL (SEQ ID NO:122) 150.000 4 390 RKFSRSDHL (SEQ ID NO:183) 150.000 5 439 QRNMTKLQL (SEQ ID NO:173) 20.000 6 329 GCNKRYFKL (SEQ ID NO:90) 10.000 7 10 ALLPAVPSL (SEQ ID NO:34) 10.000 8 180 DPMGQQGSL (SEQ ID NO:59) 9.000 9 301 RRVPGVAPT (SEQ ID NO:189) 6.000 10 126 RMFPNAPYL (SEQ ID NO:185) 5.000 11 371 KRHQRRHTG (SEQ ID NO:126) 5.000 12 225 NLYQMTSQL (SEQ ID NO:147) 5.000 13 144 IRNQGYSTV (SEQ ID NO:117) 4.000 14 429 DELVRHHNM (SEQ ID NO:53) 3.000 15 437 MHQRNMTKL (SEQ ID NO:143) 3.000 16 125 ARMFPNAPY (SEQ ID NO:38) 3.000 17 239 NQMNLGATL (SEQ ID NO:151) 3.000 18 286 YRIHTHGVF (SEQ ID NO:252) 3.000 19 174 HSFKHEDPM (SEQ ID NO:110) 3.000 20 372 RHQRRHTGV (SEQ ID NO:181) 3.000

TABLE XII Results of BIMAS HLA Peptide Binding Prediction Analysis for Binding of Human WT1 Peptides to Human HLA B40 Score (Estimate of Half Time of Disassociation of a Start Molecule Containing This Rank Position Subsequence Residue Listing Subsequence) 1 81 AEPHEEQCL (SEQ ID NO:30) 40.000 2 429 DELVRHHNM (SEQ ID NO:53) 24.000 3 410 SEKPFSCRW (SEQ ID NO:207) 20.000 4 318 SEKRPFMCA (SEQ ID NO:208) 15.000 5 233 LECMTWNQM (SEQ ID NO:131) 12.000 6 3 SDVRDLNAL (SEQ ID NO:206) 10.000 7 349 GEKPYQCDF (SEQ ID NO:91) 8.000 8 6 RDLNALLPA (SEQ ID NO:177) 5.000 9 85 EEQCLSAFT (SEQ ID NO:65) 4.000 10 315 SETSEKRPF (SEQ ID NO:209) 4.000 11 261 TEGQSNHST (SEQ ID NO:221) 4.000 12 23 GCALPVSGA (SEQ ID NO:89) 3.000 13 38 LDFAPPGAS (SEQ ID NO:130) 3.000 14 273 SDNHTTPIL (SEQ ID NO:204) 2.500 15 206 TDSCTGSQA (SEQ ID NO:220) 2.500 16 24 CALPVSGAA (SEQ ID NO:43) 2.000 17 98 GQFTGTAGA (SEQ ID NO:99) 2.000 18 30 GAAQWAPVL (SEQ ID NO:86) 2.000 19 84 HEEQCLSAF (SEQ ID NO:107) 2.000 20 26 LPVSGAAQW (SEQ ID NO:138) 2.000

TABLE XIII Results of BIMAS HLA Peptide Binding Prediction Analysis for Binding of Human WT1 Peptides to Human HLA B60 Score (Estimate of Half Time of Disassociation of a Start Molecule Containing This Rank Position Subsequence Residue Listing Subsequence) 1 81 AEPHEEQCL (SEQ ID NO:30) 160.000 2 3 SDVRDLNAL (SEQ ID NO:206) 40.000 3 429 DELVRHHNM (SEQ ID NO:53) 40.000 4 233 LECMTWNQM (SEQ ID NO:131) 22.000 5 273 SDNHTTPIL (SEQ ID NO:204) 20.000 6 209 CTGSQALLL (SEQ ID NO:52) 8.000 7 30 GAAQWAPVL (SEQ ID NO:86) 8.000 8 318 SEKRPFMCA (SEQ ID NO:208) 8.000 9 180 DPMGQQGSL (SEQ ID NO:59) 8.000 10 138 LESQPAIRN (SEQ ID NO:132) 5.280 11 239 NQMNLGATL (SEQ ID NO:151) 4.400 12 329 GCNKRYFKL (SEQ ID NO:90) 4.400 13 130 NAPYLPSCL (SEQ ID NO:144) 4.400 14 85 EEQCLSAFT (SEQ ID NO:65) 4.400 15 208 SCTGSQALL (SEQ ID NO:202) 4.000 16 207 DSCTGSQAL (SEQ ID NO:61) 4.000 17 218 RTPYSSDNL (SEQ ID NO:194) 4.000 18 261 TEGQSNHST (SEQ ID NO:221) 4.000 19 18 LGGGGGCAL (SEQ ID NO:134) 4.000 20 221 YSSDNLYQM (SEQ ID NO:253) 2.200

TABLE XIV Results of BIMAS HLA Peptide Binding Prediction Analysis for Binding of Human WT1 Peptides to Human HLA B61 Score (Estimate of Half Time of Disassociation of a Start Molecule Containing This Rank Position Subsequence Residue Listing Subsequence) 1 318 SEKRPFMCA (SEQ ID NO:208) 20.000 2 429 DELVRHHNM (SEQ ID NO:53) 16.000 3 298 QDVRRVPGV (SEQ ID NO:164) 10.000 4 81 AEPHEEQCL (SEQ ID NO:30) 8.000 5 233 LECMTWNQM (SEQ ID NO:131) 8.000 6 6 RDLNALLPA (SEQ ID NO:177) 5.500 7 85 EEQCLSAFT (SEQ ID NO:65) 4.000 8 261 TEGQSNHST (SEQ ID NO:221) 4.000 9 206 TDSCTGSQA (SEQ ID NO:220) 2.500 10 295 RGIQDVRRV (SEQ ID NO:179) 2.200 11 3 SDVRDLNAL (SEQ ID NO:206) 2.000 12 250 VAAGSSSSV (SEQ ID NO:236) 2.000 13 29 SGAAQWAPV (SEQ ID NO:211) 2.000 14 315 SETSEKRPF (SEQ ID NO:209) 1.600 15 138 LESQPAIRN (SEQ ID NO:132) 1.200 16 244 GATLKGVAA (SEQ ID NO:88) 1.100 17 20 GGGGCALPV (SEQ ID NO:92) 1.100 18 440 RNMTKLQLA (SEQ ID NO:186) 1.100 19 23 GCALPVSGA (SEQ ID NO:89) 1.100 20 191 QQYSVPPPV (SEQ ID NO:171) 1.000

TABLE XV Results of BIMAS HLA Peptide Binding Prediction Analysis for Binding of Human WT1 Peptides to Human HLA B62 Score (Estimate of Half Time of Disassociation of a Start Molecule Containing This Rank Position Subsequence Residue Listing Subsequence) 1 146 NQGYSTVTF (SEQ ID NO:150) 211.200 2 32 AQWAPVLDF (SEQ ID NO:37) 96.000 3 263 GQSNHSTGY (SEQ ID NO:100) 96.000 4 88 CLSAFTVHF (SEQ ID NO:48) 96.000 5 17 SLGGGGGCA (SEQ ID NO:215) 9.600 6 239 NQMNLGATL (SEQ ID NO:151) 8.800 7 191 QQYSVPPPV (SEQ ID NO:171) 8.000 8 98 GQFTGTAGA (SEQ ID NO:99) 8.000 9 384 QCKTCQRKF (SEQ ID NO:163) 6.000 10 40 FAPPGASAY (SEQ ID NO:74) 4.800 11 227 YQMTSQLEC (SEQ ID NO:251) 4.800 12 187 SLGEQQYSV (SEQ ID NO:214) 4.400 13 86 EQCLSAFTV (SEQ ID NO:69) 4.400 14 152 VTFDGTPSY (SEQ ID NO:244) 4.400 15 101 TGTAGACRY (SEQ ID NO:224) 4.000 16 242 NLGATLKGV (SEQ ID NO:146) 4.000 17 92 FTVHFSGQF (SEQ ID NO:85) 4.000 18 7 DLNALLPAV (SEQ ID NO:58) 4.000 19 123 GQARMFPNA (SEQ ID NO:98) 4.000 20 280 ILCGAQYRI (SEQ ID NO:116) 3.120

TABLE XVI Results of BIMAS HLA Peptide Binding Prediction Analysis for Binding of Human WT1 Peptides to Human HLA B7 Score (Estimate of Half Time of Disassociation of a Start Molecule Containing This Rank Position Subsequence Residue Listing Subsequence) 1 180 DPMGQQGSL (SEQ ID NO:59) 240.000 2 4 DVRDLNALL (SEQ ID NO:62) 200.000 3 302 RVPGVAPTL (SEQ ID NO:195) 20.000 4 30 GAAQWAPVL (SEQ ID NO:86) 12.000 5 239 NQMNLGATL (SEQ ID NO:151) 12.000 6 130 NAPYLPSCL (SEQ ID NO:144) 12.000 7 10 ALLPAVPSL (SEQ ID NO:34) 12.000 8 299 DVRRVPGVA (SEQ ID NO:63) 5.000 9 208 SCTGSQALL (SEQ ID NO:202) 4.000 10 303 VPGVAPTLV (SEQ ID NO:242) 4.000 11 18 LGGGGGCAL (SEQ ID NO:134) 4.000 12 218 RTPYSSDNL (SEQ ID NO:194) 4.000 13 207 DSCTGSQAL (SEQ ID NO:61) 4.000 14 209 CTGSQALLL (SEQ ID NO:52) 4.000 15 329 GCNKRYFKL (SEQ ID NO:90) 4.000 16 235 CMTWNQMNL (SEQ ID NO:49) 4.000 17 441 NMTKLQLAL (SEQ ID NO:149) 4.000 18 126 RMFPNAPYL (SEQ ID NO:185) 4.000 19 225 NLYQMTSQL (SEQ ID NO:147) 4.000 20 143 AIRNQGYST (SEQ ID NO:33) 3.000

TABLE XVII Results of BIMAS HLA Peptide Binding Prediction Analysis for Binding of Human WT1 Peptides to Human HLA B8 Score (Estimate of Half Time of Disassociation of a Start Molecule Containing This Rank Position Subsequence Residue Listing Subsequence) 1 329 GCNKRYFKL (SEQ ID NO:90) 16.000 2 4 DVRDLNALL (SEQ ID NO:62) 12.000 3 316 ETSEKRPFM (SEQ ID NO:73) 3.000 4 180 DPMGQQGSL (SEQ ID NO:59) 1.600 5 208 SCTGSQALL (SEQ ID NO:202) 0.800 6 130 NAPYLPSCL (SEQ ID NO:144) 0.800 7 244 GATLKGVAA (SEQ ID NO:88) 0.800 8 30 GAAQWAPVL (SEQ ID NO:86) 0.800 9 299 DVRRVPGVA (SEQ ID NO:63) 0.400 10 420 SCQKKFARS (SEQ ID NO:200) 0.400 11 387 TCQRKFSRS (SEQ ID NO:219) 0.400 12 225 NLYQMTSQL (SEQ ID NO:147) 0.400 13 141 QPAIRNQGY (SEQ ID NO:170) 0.400 14 10 ALLPAVPSL (SEQ ID NO:34) 0.400 15 207 DSCTGSQAL (SEQ ID NO:61) 0.400 16 384 QCKTCQRKF (SEQ ID NO:163) 0.400 17 136 SCLESQPAI (SEQ ID NO:198) 0.300 18 347 HTGEKPYQC (SEQ ID NO:112) 0.300 19 401 HTRTHTGKT (SEQ ID NO:114) 0.200 20 332 KRYFKLSHL (SEQ ID NO:127) 0.200

TABLE XVIII Results of BIMAS HLA Peptide Binding Prediction Analysis for Binding of Human WT1 Peptides to Human HLA B 2702 Score (Estimate of Half Time of Disassociation of a Start Molecule Containing This Rank Position Subsequence Residue Listing Subsequence) 1 332 KRYFKLSHL (SEQ ID NO:127) 900.000 2 362 RRFSRSDQL (SEQ ID NO:187) 900.000 3 286 YRIHTHGVF (SEQ ID NO:252) 200.000 4 125 ARMFPNAPY (SEQ ID NO:38) 200.000 5 375 RRHTGVKPF (SEQ ID NO:188) 180.000 6 32 AQWAPVLDF (SEQ ID NO:37) 100.000 7 301 RRVPGVAPT (SEQ ID NO:189) 60.000 8 439 QRNMTKLQL (SEQ ID NO:173) 60.000 9 126 RMFPNAPYL (SEQ ID NO:185) 22.500 10 426 ARSDELVRH (SEQ ID NO:39) 20.000 11 146 NQGYSTVTF (SEQ ID NO:150) 20.000 12 144 IRNQGYSTV (SEQ ID NO:117) 20.000 13 389 QRKFSRSDH (SEQ ID NO:172) 20.000 14 263 GQSNHSTGY (SEQ ID NO:l00) 20.000 15 416 CRWPSCQKK (SEQ ID NO:50) 20.000 16 191 QQYSVPPPV (SEQ ID NO:171) 10.000 17 217 LRTPYSSDN (SEQ ID NO:140) 10.000 18 107 CRYGPFGPP (SEQ ID NO:51) 10.000 19 98 GQFTGTAGA (SEQ ID NO:99) 10.000 20 239 NQMNLGATL (SEQ ID NO:151) 6.000

TABLE XIX Results of BIMAS HLA Peptide Binding Prediction Analysis for Binding of Human WT1 Peptides to Human HLA B 2705 Score (Estimate of Half Time of Disassociation of a Start Molecule Containing This Rank Position Subsequence Residue Listing Subsequence) 1 332 KRYFKLSHL (SEQ ID NO:127) 30000.000 2 362 RRFSRSDQL (SEQ ID NO:187) 30000.000 3 416 CRWPSCQKK (SEQ ID NO:50) 10000.000 4 439 QRNMTKLQL (SEQ ID NO:173) 2000.000 5 286 YRIHTHGVF (SEQ ID NO:252) 1000.000 6 125 ARMFPNAPY (SEQ ID NO:38) 1000.000 7 294 FRGIQDVRR (SEQ ID NO:81) 1000.000 8 432 VRHHNMHQR (SEQ ID NO:243) 1000.000 9 169 AQFPNHSFK (SEQ ID NO:36) 1000.000 10 375 RRHTGVKPF (SEQ ID NO:188) 900.000 11 126 RMFPNAPYL (SEQ ID NO:185) 750.000 12 144 IRNQGYSTV (SEQ ID NO:117) 600.000 13 301 RRVPGVAPT (SEQ ID NO:189) 600.000 14 32 AQWAPVLDF (SEQ ID NO:37) 500.000 15 191 QQYSVPPPV (SEQ ID NO:171) 300.000 16 373 HQRRHTGVK (SEQ ID NO:109) 200.000 17 426 ARSDELVRH (SEQ ID NO:39) 200.000 18 383 FQCKTCQRK (SEQ ID NO:80) 200.000 19 239 NQMNLGATL( SEQ ID NO:151) 200.000 20 389 QRKFSRSDH (SEQ ID NO:172) 200.000

TABLE XX Results of BIMAS HLA Peptide Binding Predicition Analysis for Binding of Human WT1 Peptides to Human HLA B 3501 Score (Estimate of Half Time of Disassociation of a Start Molecule Containing This Rank Position Subsequence Residue Listing Subsequence) 1 278 TPILCGAQY (SEQ ID NO:227) 40.000 2 141 QPAIRNOGY (SEQ ID NO:170) 40.000 3 219 TPYSSDNLY (SEQ ID NO:231) 40.000 4 327 YPGCNKRYF (SEQ ID NO:250) 20.000 5 163 TPSHHAAQF (SEQ ID NO:228) 20.000 6 180 DPMGQQGSL (SEQ ID NO:59) 20.000 7 221 YSSDNLYQM (SEQ ID NO:253) 20.000 8 26 LPVSGAAQW (SEQ ID NO:138) 10.000 9 174 HSFKHEDPM (SEQ ID NO:110) 10.000 10 82 EPHEEQCLS (SEQ ID NO:68) 6.000 11 213 QALLLRTPY (SEQ ID NO:160) 6.000 12 119 QASSGQARM (SEQ ID NO:161) 6.000 13 4 DVRDLNALL (SEQ ID NO:62) 6.000 14 40 FAPPGASAY (SEQ ID NO:74) 6.000 15 120 ASSGQARMF (SEQ ID NO:40) 5.000 16 207 DSCTGSQAL (SEQ ID NO:61) 5.000 17 303 VPGVAPTLV (SEQ ID NO:242) 4.000 18 316 ETSEKRPFM (SEQ ID NO:73) 4.000 19 152 VTFDGTPSY (SEQ ID NO:244) 4.000 20 412 KPFSCRWPS (SEQ ID NO:123) 4.000

TABLE XXI Results of BIMAS HLA Peptide Binding Prediction Analysis for Binding of Human WT1 Peptides to Human HLA B 3701 Score (Estimate of Half Time of Disassociation of a Molecule Start Subsequence Containing This Rank Position Residue Listing Subsequence) 1 3 SDVRDLNAL 40.000 (SEQ ID NO:206) 2 273 SDNHTTPIL 40.000 (SEQ ID NO:204) 3 81 AEPHEEQCL 10.000 (SEQ ID NO:30) 4 298 QDVRRVPGV 8.000 (SEQ ID NO:164) 5 428 SDELVRHHN 6.000 (SEQ ID NO:203) 6 85 EEQCLSAFT 5.000 (SEQ ID NO:65) 7 208 SCTGSQALL 5.000 (SEQ ID NO:202) 8 4 DVRDLNALL 5.000 (SEQ ID NO:62) 9 209 CTGSQALLL 5.000 (SEQ ID NO:52) 10 38 LDFAPPGAS 4.000 (SEQ ID NO:130) 11 223 SDNLYQMTS 4.000 (SEQ ID NO:205) 12 179 EDPMGQQGS 4.000 (SEQ ID NO:64) 13 206 TDSCTGSQA 4.000 (SEQ ID NO:220) 14 6 RDLNALLPA 4.000 (SEQ ID NO:177) 15 84 HEEQCLSAF 2.000 (SEQ ID NO:107) 16 233 LECMTWNQM 2.000 (SEQ ID NO:131) 17 429 DELVRHHNM 2.000 (SEQ ID NO:53) 18 315 SETSEKRPF 2.000 (SEQ ID NO:209) 19 349 GEKPYQCDF 2.000 (SEQ ID NO:91) 20 302 RVPGVAPTL 1.500 (SEQ ID NO:195)

TABLE XXII Results of BIMAS HLA Peptide Binding Prediction Analysis for Binding of Human WT1 Peptides to Human HLA B 3801 Score (Estimate of Half Time of Disassociation of a Molecule Start Subsequence Containing This Rank Position Residue Listing Subsequence) 1 437 MHQRNMTKL 36.000 (SEQ ID NO:143) 2 434 HHNMHQRNM 6.000 (SEQ ID NO:108) 3 372 RHQRRHTGV 6.000 (SEQ ID NO:181) 4 180 DPMGQQGSL 4.000 (SEQ ID NO:59) 5 433 RHHNMHQRN 3.900 (SEQ ID NO:180) 6 165 SHHAAQFPN 3.900 (SEQ ID NO:213) 7 202 CHTPTDSCT 3.000 (SEQ ID NO:45) 8 396 DHLKTHTRT 3.000 (SEQ ID NO:57) 9 161 GHTPSHHAA 3.000 (SEQ ID NO:94) 10 302 RVPGVAPTL 2.600 (SEQ ID NO:195) 11 417 RWPSCQKKF 2.400 (SEQ ID NO:196) 12 327 YPGCNKRYF 2.400 (SEQ ID NO:250) 13 208 SCTGSQALL 2.000 (SEQ ID NO:202) 14 163 TPSHHAAQF 2.000 (SEQ ID NO:228) 15 120 ASSGQARMF 2.000 (SEQ ID NO:40) 16 18 LGGGGGCAL 2.000 (SEQ ID NO:134) 17 177 KHEDPMGQQ 1.800 (SEQ ID NO:121) 18 83 PHEEQCLSA 1.800 (SEQ ID NO:154) 19 10 ALLPAVPSL 1.300 (SEQ ID NO:34) 20 225 NLYQMTSQL 1.300 (SEQ ID NO:147)

TABLE XXIII Results of BIMAS HLA Peptide Binding Prediction Analysis for Binding of Human WT1 Peptides to Human HLA B 3901 Score (Estimate of Half Time of Disassociation of a Molecule Start Subsequence Containing This Rank Position Residue Listing Subsequence) 1 437 MHQRNMTKL 135.000 (SEQ ID NO:143) 2 332 KRYFKLSHL 45.000 (SEQ ID NO:127) 3 434 HHNMHQRNM 30.000 (SEQ ID NO:108) 4 362 RRFSRSDQL 30.000 (SEQ ID NO:187) 5 372 RHQRRHTGV 30.000 (SEQ ID NO:181) 6 10 ALLPAVPSL 9.000 (SEQ ID NO:34) 7 439 QRNMTKLQL 7.500 (SEQ ID NO:173) 8 390 RLFSRSDHL 6.000 (SEQ ID NO:183) 9 396 DHLKTHTRT 6.000 (SEQ ID NO:57) 10 239 NQMNLGATL 6.000 (SEQ ID NO:151) 11 423 KKFARSDEL 6.000 (SEQ ID NO:122) 12 126 RMFPNAPYL 6.000 (SEQ ID NO:185) 13 225 NLYQMTSQL 6.000 (SEQ ID NO:147) 14 180 DPMGQQGSL 6.000 (SEQ ID NO:59) 15 144 IRNQGYSTV 5.000 (SEQ ID NO:117) 16 136 SCLESQPAI 4.000 (SEQ ID NO:198) 17 292 GVFRGIQDV 3.000 (SEQ ID NO:103) 18 302 RVPGVAPTL 3.000 (SEQ ID NO:195) 19 208 SCTGSQALL 3.000 (SEQ ID NO:202) 20 207 DSCTGSQAL 3.000 (SEQ ID NO:61)

TABLE XXIV Results of BIMAS HLA Peptide Binding Prediction Analysis for Binding of Human WT1 Peptides to Human HLA B 3902 Score (Estimate of Half Time of Disassociation of a Molecule Start Subsequence Containing This Rank Position Residue Listing Subsequence) 1 239 NQMNLGATL 24.000 (SEQ ID NO:151) 2 390 RKFSRSDHL 20.000 (SEQ ID NO:183) 3 423 KKFARSDEL 20.000 (SEQ ID NO:122) 4 32 AQWAPVLDF 5.000 (SEQ ID NO:37) 5 146 NQGYSTVTF 5.000 (SEQ ID NO:150) 6 130 NAPYLPSCL 2.400 (SEQ ID NO:144) 7 225 NLYQMTSQL 2.400 (SEQ ID NO:147) 8 30 GAAQWAPVL 2.400 (SEQ ID NO:86) 9 441 NMTKLQLAL 2.400 (SEQ ID NO:149) 10 302 RVPGVAPTL 2.400 (SEQ ID NO:195) 11 126 RMFPNAPYL 2.000 (SEQ ID NO:185) 12 218 RTPYSSDNL 2.000 (SEQ ID NO:194) 13 209 CTGSQALLL 2.000 (SEQ ID NO:52) 14 332 KRYFKLSHL 2.000 (SEQ ID NO:127) 15 180 DPMGQQGSL 2.000 (SEQ ID NO:59) 16 437 MHQRNMTKL 2.000 (SEQ ID NO:143) 17 207 DSCTGSQAL 2.000 (SEQ ID NO:61) 18 208 SCTGSQALL 2.000 (SEQ ID NO:202) 19 329 GCNKRYFKL 2.000 (SEQ ID NO:90) 20 10 ALLPAVPSL 2.000 (SEQ ID NO:34)

TABLE XXV Results of BIMAS HLA Peptide Binding Prediction Analysis for Binding of Human WT1 Peptides to Human HLA B 4403 Score (Estimate of Half Time of Disassociation of a Molecule Start Subsequence Containing This Rank Position Residue Listing Subsequence) 1 315 SETSEKRPF 80.000 (SEQ ID NO:209) 2 349 GEKPYQCDF 80.000 (SEQ ID NO:91) 3 84 HEEQCLSAF 60.000 (SEQ ID NO:107) 4 410 SEKPFSCRW 48.000 (SEQ ID NO:207) 5 429 DELVRHHNM 24.000 (SEQ ID NO:53) 6 278 TPILCGAQY 15.000 (SEQ ID NO:227) 7 141 QPAIRNQGY 9.000 (SEQ ID NO:170) 8 40 FAPPGASAY 9.000 (SEQ ID NO:74) 9 213 QALLLRTPY 9.000 (SEQ ID NO:160) 10 318 SEKRPFMCA 8.000 (SEQ ID NO:208) 11 81 AEPHEEQCL 8.000 (SEQ ID NO:30) 12 152 VTFDGTPSY 4.500 (SEQ ID NO:244) 13 101 TGTAGACRY 4.500 (SEQ ID NO:224) 14 120 ASSGQARMF 4.500 (SEQ ID NO:40) 15 261 TEGQSNHST 4.000 (SEQ ID NO:221) 16 85 EEQCLSAFT 4.000 (SEQ ID NO:65) 17 233 LECMTWNQM 4.000 (SEQ ID NO:131) 18 104 AGACRYGPF 4.000 (SEQ ID NO:31) 19 3 SDVRDLNAL 3.000 (SEQ ID NO:206) 20 185 QGSLGEQQY 3.000 (SEQ ID NO:166)

TABLE XXVI Results of BIMAS HLA Peptide Binding Prediction Analysis for Binding of Human WT1 Peptides to Human HLA B 5101 Score (Estimate of Half Time of Disassociation of a Molecule Start Subsequence Containing This Rank Position Residue Listing Subsequence) 1 303 VPGVAPTLV 314.600 (SEQ ID NO:242) 2 180 DPMGQQGSL 242.000 (SEQ ID NO:59) 3 250 VAAGSSSSV 157.300 (SEQ ID NO:236) 4 130 NAPYLPSCL 50.000 (SEQ ID NO:144) 5 30 GAAQWAPVL 50.000 (SEQ ID NO:86) 6 20 GGGGCALPV 44.000 (SEQ ID NO:92) 7 64 PPPPPHSFI 40.000 (SEQ ID NO:157) 8 29 SGAAQWAPV 40.000 (SEQ ID NO:211) 9 18 LGGGGGCAL 31.460 (SEQ ID NO:134) 10 295 RGIQDVRRV 22.000 (SEQ ID NO:179) 11 119 QASSGQARM 18.150 (SEQ ID NO:161) 12 418 WPSCQKKFA 12.100 (SEQ ID NO:246) 13 82 EPHEEQCLS 12.100 (SEQ ID NO:68) 14 110 GPFGPPPPS 11.000 (SEQ ID NO:96) 15 272 ESDNHTTPI 8.000 (SEQ ID NO:71) 16 306 VAPTLVRSA 7.150 (SEQ ID NO:237) 17 280 ILCGAQYRI 6.921 (SEQ ID NO:116) 18 219 TPYSSDNLY 6.600 (SEQ ID NO:231) 19 128 FPNAPYLPS 6.500 (SEQ ID NO:79) 20 204 TPTDSCTGS 6.050 (SEQ ID NO:230)

TABLE XXVII Results of BIMAS HLA Peptide Binding Prediction Analysis for Binding of Human WT1 Peptides to Human HLA B 5102 Score (Estimate of Half Time of Disassociation of a Molecule Start Subsequence Containing This Rank Position Residue Listing Subsequence) 1 295 RGIQDVRRV 290.400 (SEQ ID NO:179) 2 303 VPGVAPTLV 200.000 (SEQ ID NO:242) 3 180 DPMGQQGSL 133.100 (SEQ ID NO:59) 4 250 VAAGSSSSV 110.000 (SEQ ID NO:236) 5 30 GAAQWAPVL 55.000 (SEQ ID NO:86) 6 130 NAPYLPSCL 50.000 (SEQ ID NO:144) 7 20 GGGGCALPV 44.000 (SEQ ID NO:92) 8 29 SGAAQWAPV 44.000 (SEQ ID NO:211) 9 64 PPPPPHSFI 40.000 (SEQ ID NO:157) 10 119 QASSGQARM 36.300 (SEQ ID NO:161) 11 110 GPFGPPPPS 27.500 (SEQ ID NO:96) 12 412 KPFSCRWPS 25.000 (SEQ ID NO:123) 13 18 LGGGGGCAL 24.200 (SEQ ID NO:134) 14 24 CALPVSGAA 16.500 (SEQ ID NO:43) 15 219 TPYSSDNLY 15.000 (SEQ ID NO:231) 16 292 GVFRGIQDV 14.641 (SEQ ID NO:103) 17 136 SCLESQPAI 14.520 (SEQ ID NO:198) 18 418 WPSCQKKFA 12.100 (SEQ ID NO:246) 19 269 TGYESDNHT 11.000 (SEQ ID NO:225) 20 351 KPYQCDFKD 11.000 (SEQ ID NO:124)

TABLE XXVIII Results of BIMAS HLA Peptide Binding Prediction Analysis for Binding of Human WT1 Peptides to Human HLA B 5201 Score (Estimate of Half Time of Disassociation of a Molecule Start Subsequence Containing This Rank Position Residue Listing Subsequence) 1 191 QQYSVPPPV 100.000 (SEQ ID NO:171) 2 32 AQWAPVLDF 30.000 (SEQ ID NO:37) 3 243 LGATLKGVA 16.500 (SEQ ID NO:133) 4 303 VPGVAPTLV 13.500 (SEQ ID NO:242) 5 86 EQCLSAFTV 12.000 (SEQ ID NO:69) 6 295 RGIQDVRRV 10.000 (SEQ ID NO:179) 7 98 GQFTGTAGA 8.250 (SEQ ID NO:99) 8 292 GVFRGIQDV 8.250 (SEQ ID NO:103) 9 29 SGAAQWAPV 6.000 (SEQ ID NO:211) 10 146 NQGYSTVTF 5.500 (SEQ ID NO:150) 11 20 GGGGCALPV 5.000 (SEQ ID NO:92) 12 239 NQMNLGATL 4.000 (SEQ ID NO:151) 13 64 PPPPPHSFI 3.600 (SEQ ID NO:157) 14 273 SDNHTTPIL 3.300 (SEQ ID NO:204) 15 286 YRIHTHGVF 3.000 (SEQ ID NO:252) 16 269 TGYESDNHT 3.000 (SEQ ID NO:225) 17 406 TGKTSEKPF 2.750 (SEQ ID NO:222) 18 327 YPGCNKRYF 2.750 (SEQ ID NO:250) 19 7 DLNALLPAV 2.640 (SEQ ID NO:58) 20 104 AGACRYGPF 2.500 (SEQ ID NO:31)

TABLE XXIX Results of BIMAS HLA Peptide Binding Prediction Analysis for Binding of Human WT1 Peptides to Human HLA B 5801 Score (Estimate of Half Time of Disassociation of a Molecule Start Subsequence Containing This Rank Position Residue Listing Subsequence) 1 230 TSQLECMTW 96.800 (SEQ ID NO:234) 2 92 FTVHFSGQF 60.000 (SEQ ID NO:85) 3 120 ASSGQARMF 40.000 (SEQ ID NO:40) 4 168 AAQFPNHSF 20.000 (SEQ ID NO:29) 5 408 KTSEKPFSC 12.000 (SEQ ID NO:129) 6 394 RSDHLKTHT 9.900 (SEQ ID NO:192) 7 276 HTTPILCGA 7.200 (SEQ ID NO:115) 8 218 RTPYSSDNL 6.600 (SEQ ID NO:194) 9 152 VTFDGTPSY 6.000 (SEQ ID NO:244) 10 40 FAPPGASAY 6.000 (SEQ ID NO:74) 11 213 QALLLRTPY 4.500 (SEQ ID NO:160) 12 347 HTGEKPYQC 4.400 (SEQ ID NO:112) 13 252 AGSSSSVKW 4.400 (SEQ ID NO:32) 14 211 GSQALLLRT 4.356 (SEQ ID NO:102) 15 174 HSFKHEDPM 4.000 (SEQ ID NO:110) 16 317 TSEKRPFMC 4.000 (SEQ ID NO:233) 17 26 LPVSGAAQW 4.000 (SEQ ID NO:138) 18 289 HTHGVFRGI 3.600 (SEQ ID NO:113) 19 222 SSDNLYQMT 3.300 (SEQ ID NO:217) 20 96 FSGQFTGTA 3.300 (SEQ ID NO:82)

TABLE XXX Results of BIMAS HLA Peptide Binding Prediction Analysis for Binding of Human WT1 Peptides to Human HLA CW0301 Score (Estimate of Half Time of Disassociation of a Molecule Start Subsequence Containing This Rank Position Residue Listing Subsequence) 1 10 ALLPAVPSL 100.000 (SEQ ID NO:34) 2 332 KRYFKLSHL 48.000 (SEQ ID NO:127) 3 126 RMFPNAPYL 36.000 (SEQ ID NO:185) 4 3 SDVRDLNAL 30.000 (SEQ ID NO:206) 5 239 NQMNLGATL 24.000 (SEQ ID NO:151) 6 225 NLYQMTSQL 24.000 (SEQ ID NO:147) 7 180 DPMGQQGSL 20.000 (SEQ ID NO:59) 8 362 RRFSRSDQL 12.000 (SEQ ID NO:187) 9 329 GCNKRYFKL 10.000 (SEQ ID NO:90) 10 286 YRIHTHGVF 10.000 (SEQ ID NO:252) 11 301 RRVPGVAPT 10.000 (SEQ ID NO:189) 12 24 CALPVSGAA 10.000 (SEQ ID NO:43) 13 136 SCLESQPAI 7.500 (SEQ ID NO:198) 14 437 MHQRNMTKL 7.200 (SEQ ID NO:143) 15 390 RKFSRSDHL 6.000 (SEQ ID NO:183) 16 423 KKFARSDEL 6.000 (SEQ ID NO:122) 17 92 FTVHFSGQF 5.000 (SEQ ID NO:85) 18 429 DELVRHHNM 5.000 (SEQ ID NO:53) 19 130 NAPYLPSCL 4.800 (SEQ ID NO:144) 20 30 GAAQWAPVL 4.000 (SEQ ID NO:86)

TABLE XXXI Results of BIMAS HLA Peptide Binding Prediction Analysis for Binding of Human WT1 Peptides to Human HLA CW0401 Score (Estimate of Half Time of Disassociation of a Start Molecule Containing This Rank Position Subsequence Residue Listing Subsequence) 1 356 DFKDCERRF (SEQ ID NO:55) 120.000 2 334 YFKLSHLQM (SEQ ID NO:248) 100.000 3 180 DPMGQQGSL (SEQ ID NO:59) 88.000 4 163 TPSHHAAQF (SEQ ID NO:228) 52.800 5 327 YPGCNKRYF (SEQ ID NO:250) 40.000 6 285 QYRIHTHGV (SEQ ID NO:175) 27.500 7 424 KFARSDELV (SEQ ID NO:119) 25.000 8 326 AYPGCNKRY (SEQ ID NO:42) 25.000 9 192 QYSVPPPVY (SEQ ID NO:176) 25.000 10 417 RWPSCQKKF (SEQ ID NO:196) 22.000 11 278 TPILCGAQY (SEQ ID NO:227) 12.000 12 10 ALLPAVPSL (SEQ ID NO:34) 11.616 13 141 QPAIRNQGY (SEQ ID NO:170) 11.000 14 303 VPGVAPTLV (SEQ ID NO:242) 11.000 15 219 TPYSSDNLY (SEQ ID NO:231) 10.000 16 39 DFAPPGASA (SEQ ID NO:54) 7.920 17 99 QFTGTAGAC (SEQ ID NO:165) 6.000 18 4 DVRDLNALL (SEQ ID NO:62) 5.760 19 70 SFIKQEPSW (SEQ ID NO:210) 5.500 20 63 PPPPPPHSF (SEQ ID NO:158) 5.280

TABLE XXXII Results of BIMAS HLA Peptide Binding Prediction Analysis for Binding of Human WT1 Peptides to Human HLA CW0602 Score (Estimate of Half Time of Disassociation of a Start Molecule Containing This Rank Position Subsequence Residue Listing Subsequence) 1 332 KLRYFKLSHL (SEQ ID NO:127) 9.680 2 239 NQMNLGATL (SEQ ID NO:151) 6.600 3 130 NAPYLPSCL (SEQ ID NO:144) 6.600 4 7 DLNALLPAV (SEQ ID NO:58) 6.000 5 441 NMTKLQLAL (SEQ ID NO:149) 6.000 6 225 NLYQMTSQL (SEQ ID NO:147) 6.000 7 4 DVRDLNALL (SEQ ID NO:62) 6.000 8 3 SDVRDLNAL (SEQ ID NO:206) 4.400 9 10 ALLPAVPSL (SEQ ID NO:34) 4.000 10 213 QALLLRTPY (SEQ ID NO:160) 3.300 11 319 EKRPFMCAY (SEQ ID NO:67) 3.000 12 30 GAAQWAPVL (SEQ ID NO:86) 2.200 13 242 NLGATLKGV (SEQ ID NO:146) 2.200 14 292 GVFRGIQDV (SEQ ID NO:103) 2.200 15 207 DSCTGSQAL (SEQ ID NO:61) 2.200 16 362 RRFSRSDQL (SEQ ID NO:187) 2.200 17 439 QRNMTKLQL (SEQ ID NO:173) 2.200 18 295 RGIQDVRRV (SEQ ID NO:179) 2.200 19 423 KKFARSDEL (SEQ ID NO:122) 2.200 20 180 DPMGQQGSL (SEQ ID NO:59) 2.200

TABLE XXXIII Results of BIMAS HLA Peptide Binding Prediction Analysis for Binding of Human WT1 Peptides to Human HLA CW0702 Score (Estimate of Half Time of Disassociation of a Start Molecule Containing This Rank Position Subsequence Residue Listing Subsequence 1 319 EKRPFMCAY (SEQ ID NO:67) 26.880 2 326 AYPGCNKRY (SEQ ID NO:42) 24.000 3 40 FAPPGASAY (SEQ ID NO:74) 14.784 4 192 QYSVPPPVY (SEQ ID NO:176) 12.000 5 278 TPILCGAQY (SEQ ID NO:227) 12.000 6 219 TPYSSDNLY (SEQ ID NO:231) 12.000 7 213 QALLLRTPY (SEQ ID NO:160) 8.800 8 125 ARMFPNAPY (SEQ ID NO:38) 8.000 9 327 YPGCNKRYF (SEQ ID NO:250) 6.600 10 152 VTFDGTPSY (SEQ ID NO:244) 5.600 11 141 QPAIRNQGY (SEQ ID NO:170) 4.800 12 345 RKHTGEKPY (SEQ ID NO:184) 4.000 13 185 QGSLGEQQY (SEQ ID NO:166) 4.000 14 101 TGTAGACRY (SEQ ID NO:224) 4.000 15 375 RRHTGVKPF (SEQ ID NO:188) 4.000 16 263 GQSNHSTGY (SEQ ID NO:100) 4.000 17 163 TPSHHAAQF (SEQ ID NO:228) 3.000 18 33 QWAPVLDFA (SEQ ID NO:174) 2.688 19 130 NAPYLPSCL (SEQ ID NO:144) 2.640 20 84 HEEQCLSAF (SEQ ID NO:107) 2.400

TABLE XXXIV Results of BIMAS HLA Peptide Binding Prediction Analysis for Binding of Human WT1 Peptides to Mouse MHC Class I Db Score (Estimate of Half Time of Disassociation of a Start Molecule Containing This Rank Position Subsequence Residue Listing Subsequence) 1 235 CMTWNQMNL (SEQ ID NO:49) 5255.712 2 126 RMFPNAPYL (SEQ ID NO:185) 1990.800 3 221 YSSDNLYQM (SEQ ID NO:253) 930.000 4 228 QMTSQLECM (SEQ ID NO:169) 33.701 5 239 NQMNLGATL (SEQ ID NO:151) 21.470 6 441 NMTKLQLAL (SEQ ID NO:149) 19.908 7 437 MHQRNMTKL (SEQ ID NO:143) 19.837 8 136 SCLESQPAI (SEQ ID NO:198) 11.177 9 174 HSFKHEDPM (SEQ ID NO:110) 10.800 10 302 RVPGVAPTL (SEQ ID NO:195) 10.088 11 130 NAPYLPSCL (SEQ ID NO:144) 8.400 12 10 ALLPAVPSL (SEQ ID NO:34) 5.988 13 208 SCTGSQALL (SEQ ID NO:202) 4.435 14 209 CTGSQALLL (SEQ ID NO:52) 3.548 15 238 WNQMNLGAT (SEQ ID NO:245) 3.300 16 218 RTPYSSDNL (SEQ ID NO:194) 3.185 17 24 CALPVSGAA (SEQ ID NO:43) 2.851 18 18 LGGGGGCAL (SEQ ID NO:134) 2.177 19 142 PAIRNQGYS (SEQ ID NO:152) 2.160 20 30 GAAQWAPVL (SEQ ID NO:86) 1.680

TABLE XXXV Results of BIMAS HLA Peptide Binding Prediction Analysis for Binding of Human WT1 Peptides to Mouse MHC Class I Dd Score (Estimate of Half Time of Disassociation of a Start Molecule Containing This Rank Position Subsequence Residue Listing Subsequence) 1 112 FGPPPPSQA (SEQ ID NO:76) 48.000 2 122 SGQARMFPN (SEQ ID NO:212) 36.000 3 104 AGACRYGPF (SEQ ID NO:31) 30.000 4 218 RTPYSSDNL (SEQ ID NO:194) 28.800 5 130 NAPYLPSCL (SEQ ID NO:144) 20.000 6 302 RVPGVAPTL (SEQ ID NO:195) 20.000 7 18 LGGGGGCAL (SEQ ID NO:134) 20.000 8 81 AEPHEEQCL (SEQ ID NO:30) 10.000 9 29 SGAAQWAPV (SEQ ID NO:211) 7.200 10 423 KKFARSDEL (SEQ ID NO:122) 7.200 11 295 RGIQDVRRV (SEQ ID NO:179) 7.200 12 390 RKFSRSDHL (SEQ ID NO:183) 6.000 13 332 KRYFKLSHL (SEQ ID NO:127) 6.000 14 362 RRFSRSDQL (SEQ ID NO:187) 6.000 15 417 RWPSCQKKF (SEQ ID NO:196) 6.000 16 160 YGHTPSHHA (SEQ ID NO:249) 6.000 17 20 GGGGCALPV (SEQ ID NO:92) 6.000 18 329 GCNKRYFKL (SEQ ID NO:90) 5.000 19 372 RHQRRHTGV (SEQ ID NO:181) 4.500 20 52 GGPAPPPAP (SEQ ID NO:93) 4.000

TABLE XXXVI Results of BIMAS HLA Peptide Binding Prediction Analysis for Binding of Human WT1 Peptides to Mouse MHC Class I Kb Score (Estimate of Half Time of Disassociation of a Start Molecule Containing This Rank Position Subsequence Residue Listing Subsequence) 1 329 GCNKRYFKL (SEQ ID NO:90) 24.000 2 225 NLYQMTSQL (SEQ ID NO:147) 10.000 3 420 SCQKKFARS (SEQ ID NO:200) 3.960 4 218 RTPYSSDNL (SEQ ID NO:194) 3.630 5 437 MHQRNMTKL (SEQ ID NO:143) 3.600 6 387 TCQRKFSRS (SEQ ID NO:219) 3.600 7 302 RVPGVAPTL (SEQ ID NO:195) 3.300 8 130 NAPYLPSCL (SEQ ID NO:144) 3.000 9 289 HTHGVFRGI (SEQ ID NO:113) 3.000 10 43 PGASAYGSL (SEQ ID NO:153) 2.400 11 155 DGTPSYGHT (SEQ ID NO:56) 2.400 12 273 SDNHTTPIL (SEQ ID NO:204) 2.200 13 126 RMFPNAPYL (SEQ ID NO:185) 2.200 14 128 FPNAPYLPS (SEQ ID NO:79) 2.000 15 3 SDVRDLNAL (SEQ ID NO:206) 1.584 16 207 DSCTGSQAL (SEQ ID NO:61) 1.584 17 332 KRYFKLSHL (SEQ ID NO:127) 1.500 18 18 LGGGGGCAL (SEQ ID NO:134) 1.320 19 233 LECMTWNQM (SEQ ID NO:131) 1.320 20 441 NMTKLQLAL (SEQ ID NO:149) 1.200

TABLE XXXVII Results of BIMAS HLA Peptide Binding Prediction Analysis for Binding of Human WT1 Peptides to Mouse MHC Class I Kd Score (Estimate of Half Time of Disassociation of a Start Molecule Containing This Rank Position Subsequence Residue Listing Subsequence) 1 285 QYRIHTHGV (SEQ ID NO:175) 600.000 2 424 KFARSDELV (SEQ ID NO:119) 288.000 3 334 YFKLSHLQM (SEQ ID NO:248) 120.000 4 136 SCLESQPTI (SEQ ID NO:199) 115.200 5 239 NQMNLGATL (SEQ ID NO:151) 115.200 6 10 ALLPAVSSL (SEQ ID NO:35) 115.200 7 47 AYGSLGGPA (SEQ ID NO:41) 86.400 8 180 DPMGQQGSL (SEQ ID NO:59) 80.000 9 270 GYESDNHTA (SEQ ID NO:105) 72.000 10 326 AYPGCNKRY (SEQ ID NO:42) 60.000 11 192 QYSVPPPVY (SEQ ID NO:176) 60.000 12 272 ESDNHTAPI (SEQ ID NO:70) 57.600 13 289 HTHGVFRGI (SEQ ID NO:113) 57.600 14 126 DVRDLNALL (SEQ ID NO:62) 57.600 15 4 CTGSQALLL (SEQ ID NO:52) 57.600 16 208 SCTGSQALL (SEQ ID NO:202) 48.000 17 441 NMTKLQLAL (SEQ ID NO:149) 48.000 18 207 DSCTGSQAL (SEQ ID NO:61) 48.000 19 130 NAPYLPSCL (SEQ ID NO:144) 48.000 20 235 CMTWNQMNL (SEQ ID NO:49) 48.000

TABLE XXXVIII Results of BIMAS HLA Peptide Binding Prediction Analysis for Binding of Human WT1 Peptides to Mouse MHC Class I Kk Score (Estimate of Half Time of Disassociation of a Start Molecule Containing This Rank Position Subsequence Residue Listing Subsequence) 1 81 AEPHEEQCL (SEQ ID NO:30) 40.000 2 85 EEQCLSAFT (SEQ ID NO:65) 40.000 3 429 DELVRHHNM (SEQ ID NO:53) 20.000 4 315 SETSEKRPF (SEQ ID NO:209) 20.000 5 261 TEGQSNHST (SEQ ID NO:221) 20.000 6 410 SEKPFSCRW (SEQ ID NO:207) 10.000 7 272 ESDNHTTPI (SEQ ID NO:71) 10.000 8 318 SEKRPFMCA (SEQ ID NO:208) 10.000 9 138 LESQPAIRN (SEQ ID NO:132) 10.000 10 233 LECMTWNQM (SEQ ID NO:131) 10.000 11 298 QDVRRVPGV (SEQ ID NO:164) 10.000 12 84 HEEQCLSAF (SEQ ID NO:107) 10.000 13 349 GEKPYQCDF (SEQ ID NO:91) 10.000 14 289 HTHGVFRGI (SEQ ID NO:113) 10.000 15 179 EDPMGQQGS (SEQ ID NO:64) 8.000 16 136 SCLESQPAI (SEQ ID NO:198) 5.000 17 280 ILCGAQYRI (SEQ ID NO:116) 5.000 18 273 SDNHTTPIL (SEQ ID NO:204) 5.000 19 428 SDELVRHHN (SEQ ID NO:203) 4.000 20 3 SDVRDLNAL (SEQ ID NO:206) 4.000

TABLE XXXIX Results of BIMAS HLA Peptide Binding Prediction Analysis for Binding of Human WT1 Peptides to Mouse MHC Class I Ld Score (Estimate of Half Time of Disassociation of a Start Molecule Containing This Rank Position Subsequence Residue Listing Subsequence) 1 163 TPSHHAAQF (SEQ ID NO:228) 360.000 2 327 YPGCNKRYF (SEQ ID NO:250) 300.000 3 180 DPMGQQGSL (SEQ ID NO:59) 150.000 4 26 LPVSGAAQW (SEQ ID NO:138) 93.600 5 278 TPILCGAQY (SEQ ID NO:227) 72.000 6 141 QPAIRNQGY (SEQ ID NO:170) 60.000 7 219 TPYSSDNLY (SEQ ID NO:231) 60.000 8 303 VPGVAPTLV (SEQ ID NO:242) 60.000 9 120 ASSGQARMF (SEQ ID NO:40) 50.000 10 63 PPPPPPHSF (SEQ ID NO:158) 45.000 11 113 GPPPPSQAS (SEQ ID NO:97) 45.000 12 157 TPSYGHTPS (SEQ ID NO:229) 39.000 13 207 DSCTGSQAL (SEQ ID NO:61) 32.500 14 110 GPFGPPPPS (SEQ ID NO:96) 30.000 15 82 EPHEEQCLS (SEQ ID NO:68) 30.000 16 412 KPFSCRWPS (SEQ ID NO:123) 30.000 17 418 WPSCQKKFA (SEQ ID NO:246) 30.000 18 221 YSSDNLYQM (SEQ ID NO:253) 30.000 19 204 TPTDSCTGS (SEQ ID NO:230) 30.000 20 128 FPNAPYLPS (SEQ ID NO:79) 30.000

TABLE XL Results of BIMAS HLA Peptide Binding Prediction Analysis for Binding of Human WT1 Peptides to Cattle HLA A20 Score (Estimate of Half Time of Disassociation of a Start Molecule Containing This Rank Position Subsequence Residue Listing Subsequence) 1 350 EKPYQCDFK (SEQ ID NO:66) 1000.00 2 319 EKRPFMCAY (SEQ ID NO:67) 500.000 3 423 KKFARSDEL (SEQ ID NO:122) 500.000 4 345 RKHTGEKPY (SEQ ID NO:184) 500.000 5 390 RKFSRSDHL (SEQ ID NO:183) 500.000 6 137 CLESQPAIR (SEQ ID NO:47) 120.000 7 380 VKPFQCKTC (SEQ ID NO:239) 100.000 8 407 GKTSEKPFS (SEQ ID NO:95) 100.000 9 335 FKLSHLQMH (SEQ ID NO:78) 100.000 10 247 LKGVAAGSS (SEQ ID NO:135) 100.000 11 370 LKRHQRRHT (SEQ ID NO:136) 100.000 12 258 VKWTEGQSN (SEQ ID NO:240) 100.000 13 398 LKTHTRTHT (SEQ ID NO:137) 100.000 14 331 NKRYFKLSH (SEQ ID NO:145) 100.000 15 357 FKDCERRFS (SEQ ID NO:77) 100.000 16 385 CKTCQRKFS (SEQ ID NO:46) 100.000 17 294 FRGIQDVRR (SEQ ID NO:81) 80.000 18 368 DQLKRHQRR (SEQ ID NO:60) 80.000 19 432 VRHHNMHQR (SEQ ID NO:243) 80.000 20 118 SQASSGQAR (SEQ ID NO:216) 80.000

TABLE XLI Results of BIMAS HLA Peptide Binding Prediction Analysis for Binding of Mouse WT1 Peptides to Mouse MHC Class I A 0201 Score (Estimate of Half Time of Disassociation of a Start Molecule Containing This Rank Position Subsequence Residue Listing Subsequence) 1 126 RMFPNAPYL (SEQ ID NO:293) 313.968 2 187 SLGEQQYSV (SEQ ID NO:299) 285.163 3 10 ALLPAVSSL (SEQ ID NO:255) 181.794 4 225 NLYQMTSQL (SEQ ID NO:284) 68.360 5 292 GVFRGIQDV (SEQ ID NO:270) 51.790 6 93 TLHFSGQFT (SEQ ID NO:302) 40.986 7 191 QQYSVPPPV (SEQ ID NO:290) 22.566 8 280 ILCGAQYRI (SEQ ID NO:274) 17.736 9 441 NMTKLHVAL (SEQ ID NO:285) 15.428 10 235 CMTWNQMNL (SEQ ID NO:258) 15.428 11 7 DLNALLPAV (SEQ ID NO:261) 11.998 12 242 NLGATLKGM (SEQ ID NO:283) 11.426 13 227 YQMTSQLEC (SEQ ID NO:307) 8.573 14 239 NQMNLGATL (SEQ ID NO:286) 8.014 15 309 TLVRSASET (SEQ ID NO:303) 7.452 16 408 KTSEKPFSC (SEQ ID NO:277) 5.743 17 340 LQMHSRKHT (SEQ ID NO:280) 4.752 18 228 QMTSQLECM (SEQ ID NO:289) 4.044 19 37 VLDFAPPGA (SEQ ID NO:304) 3.378 20 302 RVSGVAPTL (SEQ ID NO:295) 1.869

TABLE XLII Results of BIMAS HLA Peptide Binding Prediction Analysis for Binding of Mouse WT1 Peptides to Mouse MHC Class I Db Score (Estimate of Half Time of Disassociation of a Start Molecule Containing This Rank Position Subsequence Residue Listing Subsequence) 1 221 YSSDNLYQM (SEQ ID NO:308) 312.000 2 126 RMFPNAPYL (SEQ ID NO:293) 260.000 3 235 CMTWNQMNL (SEQ ID NO:258) 260.000 4 437 MHQRNMTKL (SEQ ID NO:281) 200.000 5 238 WNQMNLGAT (SEQ ID NO:305) 12.000 6 130 NAPYLPSCL (SEQ ID NO:282) 8.580 7 3 SDVRDLNAL (SEQ ID NO:298) 7.920 8 136 SCLESQPTI (SEQ ID NO:296) 7.920 9 81 AEPHEEQCL (SEQ ID NO:254) 6.600 10 10 ALLPAVSSL (SEQ ID NO:255) 6.600 11 218 RTPYSSDNL (SEQ ID NO:294) 6.000 12 441 NMTKLHVAL (SEQ ID NO:285) 3.432 13 228 QMTSQLECM (SEQ ID NO:289) 3.120 14 174 HSFKHEDPM (SEQ ID NO:272) 3.120 15 242 NLGATLKGM (SEQ ID NO:283) 2.640 16 261 TEGQSNHGI (SEQ ID NO:301) 2.640 17 225 NLYQMTSQL (SEQ ID NO:284) 2.640 18 207 DSCTGSQAL (SEQ ID NO:263) 2.600 19 119 QASSGQARM (SEQ ID NO:288) 2.600 20 18 LGGGGGCGL (SEQ ID NO:279) 2.600

TABLE XLIII Results of BIMAS HLA Peptide Binding Prediction Analysis for Binding of Mouse WT1 Peptides to Mouse MHC Class I Kb Score (Estimate of Half Time of Disassociation of a Start Molecule Containing This Rank Position Subsequence Residue Listing Subsequence) 1 329 GCNKRYFKL (SEQ ID NO:268) 24.000 2 225 NLYQMTSQL (SEQ ID NO:284) 10.000 3 420 SCQKKFARS (SEQ ID NO:297) 3.960 4 218 RTPYSSDNL (SEQ ID NO:294) 3.630 5 437 MHQRNMTKL (SEQ ID NO:281) 3.600 6 387 TCQRKFSRS (SEQ ID NO:300) 3.600 7 289 HTHGVFRGI (SEQ ID NO:273) 3.000 8 130 NAPYLPSCL (SEQ ID NO:282) 3.000 9 43 PGASAYGSL (SEQ ID NO:287) 2.400 10 155 DGAPSYGHT (SEQ ID NO:260) 2.400 11 126 RMFPNAPYL (SEQ ID NO:293) 2.200 12 128 FPNAPYLPS (SEQ ID NO:267) 2.000 13 207 DSCTGSQAL (SEQ ID NO:263) 1.584 14 3 SDVRDLNAL (SEQ ID NO:298) 1.584 15 332 KRYFKLSHL (SEQ ID NO:276) 1.500 16 233 LECMTWNQM (SEQ ID NO:278) 1.320 17 18 LGGGGGCGL (SEQ ID NO:279) 1.320 18 242 NLGATLKGM (SEQ ID NO:283) 1.200 19 123 GQARMFPN (SEQ ID NO:269)A 1.200 20 441 NMTKLHVAL (SEQ ID NO:285) 1.200

TABLE XLIV Results of BIMAS HLA Peptide Binding Prediction Analysis for Binding of Mouse WT1 Peptides to Mouse MHC Class I Kd Score (Estimate of Half Time of Disassociation of a Start Molecule Containing This Rank Position Subsequence Residue Listing Subsequence) 1 285 QYRIHTHGV (SEQ ID NO:291) 600.000 2 424 KFARSDELV (SEQ ID NO:275) 288.000 3 334 YFKLSHLQM (SEQ ID NO:306) 120.000 4 136 SCLESQPTI (SEQ ID NO:296) 115.200 5 239 NQMNLGATL (SEQ ID NO:286) 115.200 6 10 ALLPAVSSL (SEQ ID NO:255) 115.200 7 47 AYGSLGGPA (SEQ ID NO:256) 86.400 8 180 DPMGQQGSL (SEQ ID NO:262) 80.000 9 270 GYESDNHTA (SEQ ID NO:271) 72.000 10 192 QYSVPPPVY (SEQ ID NO:292) 60.000 11 326 AYPGCNKRY (SEQ ID NO:257) 60.000 12 289 HTHGVFRGI (SEQ ID NO:273) 57.600 13 4 DVRDLNALL (SEQ ID NO:264) 57.600 14 126 RMFPNAPYL (SEQ ID NO:293) 57.600 15 209 CTGSQALLL (SEQ ID NO:259) 48.000 16 86 EQCLSAFTL (SEQ ID NO:265) 48.000 17 302 RVSGVAPTL (SEQ ID NO:295) 48.000 18 218 RTPYSSDNL (SEQ ID NO:294) 48.000 19 272 ESDNHTAPI (SEQ ID NO:266) 48.000 20 225 NLYQMTSQL (SEQ ID NO:284) 48.000

TABLE XLV Results of TSites Peptide Binding Prediction Ana- lysis for Human WT1 Peptides Capable of Eliciting a Helper T cell Response Peptide Sequence  p6–23 RDLNALLPAVPSLGGGG (SEQ ID NO:1) p30–35 GAAQWA (SEQ ID NO:309) p45–56 ASAYGSLGGPAP (SEQ ID NO:310) p91–105 AFTVHFSGQFTGTAG (SEQ ID NO:311) p117–139 PSQASSGQARMFPNAPYLPSCLE (SEQ ID NO:2) p167–171 HAAQF (SEQ ID NO:312) p202–233 CHTPTDSCTGSQALLLRTPYSSDNLYQ (SEQ ID NO:313) MTSQL p244–262 GATLKGVAAGSSSSVKWTE (SEQ ID NO:4) p287–318 RIHTHGVFRGIQDVRRVPGVAPTLVRS (SEQ ID NO:314) ASETS p333–336 RYFK (SEQ ID NO:315) p361–374 ERRFSRSDQLKRHQ (SEQ ID NO:316) p389–4l0 QRKFSRSDHLKTHTRTHTGKTS (SEQ ID NO:317) p421–441 CQKKFARSDELVRHHNMHQRN (SEQ ID NO:318)

Certain CTL peptides (shown in Table XLVI) were selected for further study. For each peptide in Table XLVI, scores obtained using BIMAS HLA peptide binding prediction analysis are provided.

TABLE XLVI WT1 Peptide Sequences and HLA Peptide Binding Predictions Peptide Sequence Comments p329–337 GCNKRYFKL (SEQ ID Nos:90 and 268) Score 24,000 p225–233 NLYQMTSQL (SEQ ID Nos:147 and 284) binds also to class II and HLA A2, Kd, score 10,000 p235–243 CMTWNQMNL (SEQ ID Nos:49 and 258) binds also to HLA A2, score 5,255,712 p126–I34 RMFPNAPYL (SEQ ID Nos:185 and 293) binds also to Kd, class II and HLA A2, score 1,990,800 p221–229 YSSDNLYQM (SEQ ID Nos:253 and 308) binds also to Ld, score 312,000 p228–236 QMTSQLECM (SEQ ID Nos:169 and 289) score 3,120 p239–247 NQMNLGATL (SEQ ID Nos:151 and 286) binds also to HLA A 0201, Kd, score 8,015 mouse p136–144 SCLESQPTI (SEQ ID NO:296) binds also to Kd, 1mismatch to human human p136–144 SCLESQPAI (SEQ ID NO:198) score 7,920 mouse p10–18 ALLPAVSSL (SEQ ID NO:255) binds also to Kd, HLA A2, 1 mismatch to human human p10–18 ALLPAVPSL (SEQ ID NO:34) score 6,600

Peptide binding to C57B1/6 murine MHC was confirmed using the leukemia cell line RMA-S, as described by Ljunggren et al., Nature 346:476–480, 1990. In brief, RMA-S cells were cultured for 7 hours at 26° C. in complete medium supplemented with 1% FCS. A total of 10⁶ RMA-S cells were added into each well of a 24-well plate and incubated either alone or with the designated peptide (25 ug/ml) for 16 hours at 26° C. and additional 3 hours at 37° C. in complete medium. Cells were then washed three times and stained with fluorescein isothiocyanate-conjugated anti D^(b) or anti-K^(b) antibody (PharMingen, San Diego, Calif.). Labeled cells were washed twice, resuspended and fixed in 500 ul of PBS with 1% paraformaldehyde and analyzed for fluorescence intensity in a flow cytometer (Becton-Dickinson FACSCalibur®). The percentage of increase of D^(b) or K^(b) molecules on the surface of the RMA-S cells was measured by increased mean fluorescent intensity of cells incubated with peptide compared with that of cells incubated in medium alone.

Mice were immunized with the peptides capable of binding to murine class I MHC. Following immunization, spleen cells were stimulated in vitro and tested for the ability to lyse targets incubated with WT1 peptides. CTL were evaluated with a standard chromium release assay (Chen et al., Cancer Res. 54:1065–1070, 1994). 106 target cells were incubated at 37° C. with 150 μCi of sodium ⁵¹Cr for 90 minutes, in the presence or absence of specific peptides. Cells were washed three times and resuspended in RPMI with 5% fetal bovine serum. For the assay, 10⁴ ⁵¹Cr-labeled target cells were incubated with different concentrations of effector cells in a final volume of 200 μl in U-bottomed 96-well plates. Supernatants were removed after 4 to 7 hours at 37° C., and the percentage specific lysis was determined by the formula: % specific lysis=100×(experimental release−spontaneous release)/(maximum release−spontaneous release).

The results, presented in Table XLVII, show that some WT1 peptides can bind to class I MHC molecules, which is essential for generating CTL. Moreover, several of the peptides were able to elicit peptide specific CTL (FIGS. 9A and 9B), as determined using chromium release assays. Following immunization to CTL peptides p10–18 human, p136–144 human, p136–144 mouse and p235–243, peptide specific CTL lines were generated and clones were established. These results indicate that peptide specific CTL can kill malignant cells expressing WT1.

TABLE XLVII Binding of WT1 CTL Peptides to mouse B6 class I antigens Peptide Binding Affinity to Mouse MUC Class I Positive control 91% negative control  0.5.–1.3% p235–243 33.6% p136–144 mouse 27.9% p136–144 human 52% p10–18: human  2.2% p225–233  5.8% p329–337  1.2% p126–134  0.9% p221–229  0.8% p228–236  1.2% p239–247  1%

Example 5 Use of a WT1 Polypeptide to Elicit WT1 Specific CTL in Mice

This Example illustrates the ability of a representative WT1 polypeptide to elicit CTL immunity capable of killing WT1 positive tumor cell lines.

P117–139, a peptide with motifs appropriate for binding to class I and class II MHC, was identified as described above using TSITES and BIMAS HLA peptide binding prediction analyses. Mice were immunized as described in Example 3. Following immunization, spleen cells were stimulated in vitro and tested for the ability to lyse targets incubated with WT1 peptides, as well as WT1 positive and negative tumor cells. CTL were evaluated with a standard chromium release assay. The results, presented in FIGS. 10A–10D, show that P117 can elicit WT1 specific CTL capable of killing WT1 positive tumor cells, whereas no killing of WT1 negative cells was observed. These results demonstrate that peptide specific CTL in fact kill malignant cells expressing WT1 and that vaccine and T cell therapy are effective against malignancies that express WT1.

Similar immunizations were performed using the 9-mer class I MHC binding peptides p136–144, p225–233, p235–243 as well as the 23-mer peptide p117–139. Following immunization, spleen cells were stimulated in vitro with each of the 4 peptides and tested for ability to lyse targets incubated with WT1 peptides. CTL were generated specific for p136–144, p235–243 and p117–139, but not for p225–233. CTL data for p235–243 and p117–139 are presented in FIGS. 11A and 11B. Data for peptides p136–144 and p225–233 are not depicted.

CTL lysis demands that the target WT1 peptides are endogenously processed and presented in association with tumor cell class I MHC molecules. The above WT1 peptide specific CTL were tested for ability to lyse WT1 positive versus negative tumor cell lines. CTL specific for p235–243 lysed targets incubated with the p235–243 peptides, but failed to lyse cell lines that expressed WT1 proteins (FIG. 11A). By marked contrast, CTL specific for p117–139 lysed targets incubated with p117–139 peptides and also lysed malignant cells expressing WT1 (FIG. 11B). As a negative control, CTL specific for p117–139 did not lyse WT1 negative EL-4 (also referred to herein as E10).

Specificity of WT1 specific lysis was confirmed by cold target inhibition (FIGS. 12A–12B). Effector cells were plated for various effector: target ratios in 96-well U-bottom plates. A ten-fold excess (compared to hot target) of the indicated peptide-coated target without ⁵¹Cr labeling was added. Finally, 10⁴ ⁵¹Cr-labeled target cells per well were added and the plates incubated at 37° C. for 4 hours. The total volume per well was 200 μl .

Lysis of TRAMP-C by p117–139 specific CTL was blocked from 58% to 36% by EL-4 incubated with the relevant peptide p117–139, but not with EL-4 incubated with an irrelevant peptide (FIG. 12A). Similarly, lysis of BLK-SV40 was blocked from 18% to 0% by EL-4 incubated with the relevant peptide p117–139 (FIG. 12B). Results validate that WT1 peptide specific CTL specifically kill malignant cells by recognition of processed WT1.

Several segments with putative CTL motifs are contained within p117–139. To determine the precise sequence of the CTL epitope all potential 9-mer peptides within p117–139 were synthesized (Table XLVIII). Two of these peptides (p126–134 and p130–138) were shown to bind to H-2b class I molecules (Table XLVIII). CTL generated by immunization with p117–139 lysed targets incubated with p126–134 and p130–138, but not the other 9-mer peptides within p117–139 (FIG. 13A).

The p117–139 specific CTL line was restimulated with either p126–134 or p130–138. Following restimulation with p126–134 or p130–138, both T cell lines demonstrated peptide specific lysis, but only p130–138 specific CTL showed lysis of a WT1 positive tumor cell line (FIGS. 13B and 13C). Thus, p130–138 appears to be the naturally processed epitope.

TABLE XLVIII Binding of WT1 CTL 9mer Peptides within p117–139 to mouse B6 class I antigens Bind- ing Affin- ity to Mouse MHC Peptide Class I P117–125 PSQASSGQA (SEQ ID NO:221) 2% P118–126 SQASSGQAR (SEQ ID NO:216) 2% P119–127 QASSGQARM (SEQ ID Nos:161 and 288) 2% P120–128 ASSGQARMF (SEQ ID NO:40) 1% P121–129 SSGQARMFP (SEQ ID NO:222) 1% P122–130 SGQARMFPN (SEQ ID NO:212) 1% P123–131 GQARMFPNA (SEQ ID Nos:98 and 269) 1% P124–132 QARMFPNAP (SEQ ID NO:223) 1% P125–133 APMFPNAPY (SEQ ID NO:38) 1% P126–134 RMFPNAPYL (SEQ ID Nos:185 and 293) 79% P127–135 MFPNAPYLP (SEQ ID NO:224) 2% P128–136 FPNAPYLPS (SEQ ID Nos:79 and 267) 1% P129–137 PNAPYLPSC (SEQ ID NO:225) 1% P130–138 NAPYLPSCL (SEQ ID Nos:144 and 282) 79% P131–139 APYLPSCLE (SEQ ID NO:226) 1%

Example 6 Identification of WT1 Specific mRNA in Mouse Tumor Cell Lines

This Example illustrates the use of RT-PCR to detect WT1 specific mRNA in cells and cell lines.

Mononuclear cells were isolated by density gradient centrifugation, and were immediately frozen and stored at −80° C. until analyzed by RT-PCR for the presence of WT1 specific mRNA. RT-PCR was generally performed as described by Fraizer et al., Blood 86:4704–4706, 1995. Total RNA was extracted from 10⁷ cells according to standard procedures. RNA pellets were resuspended in 25 μL diethylpyrocarbonate treated water and used directly for reverse transcription. The zinc-finger region (exons 7 to 10) was amplified by PCR as a 330 bp mouse cDNA. Amplification was performed in a thermocycler during one or, when necessary, two sequential rounds of PCR. AmpliTaq DNA Polymerase (Perkin Elmer Cetus, Norwalk, Conn.), 2.5 mM MgCl₂ and 20 pmol of each primer in a total reaction volume of 50 μl were used. Twenty μL aliquots of the PCR products were electrophoresed on 2% agarose gels stained with ethidium bromide. The gels were photographed with Polaroid film (Polaroid 667, Polaroid Ltd., Hertfordshire, England). Precautions against cross contamination were taken following the recommendations of Kwok and Higuchi, Nature 339:237–238, 1989. Negative controls included the cDNA- and PCR-reagent mixes with water instead of cDNA in each experiment. To avoid false negatives, the presence of intact RNA and adequate cDNA generation was evaluated for each sample by a control PCR using β-actin primers. Samples that did not amplify with these primers were excluded from analysis.

Primers for amplification of WT1 in mouse cell lines were: P115: 1458–1478: 5′ CCC AGG CTG CAA TAA GAG ATA 3′ (forward primer; SEQ ID NO:21); and P116: 1767–1787: 5′ ATG TTG TGA TGG CGG ACC AAT 3′ (reverse primer; SEQ ID NO:22) (see Inoue et al, Blood 88:2267–2278, 1996; Fraizer et al., Blood 86:4704–4706, 1995).

Beta Actin primers used in the control reactions were: 5′ GTG GGG CGC CCC AGG CAC CA 3′ (sense primer; SEQ ID NO:23); and 5′ GTC CTT AAT GTC ACG CAC GAT TTC 3′ (antisense primer; SEQ ID NO:24)

Primers for use in amplifying human WT1 include: P117: 954–974: 5′ GGC ATC TGA GAC CAG TGA GAA 3′ (SEQ ID NO:25); and P118: 1434–1414: 5′ GAG AGT CAG ACT TGA AAG CAGT 3′ (SEQ ID NO:5). For nested RT-PCR, primers may be: P119: 1023–1043: 5′ GCT GTC CCA CTT ACA GAT GCA 3′ (SEQ ID NO:26); and P120: 1345–1365: 5′ TCA AAG CGC CAG CTG GAG TTT 3′ (SEQ ID NO:27).

Table XLVIII shows the results of WT1 PCR analysis of mouse tumor cell lines. Within Table IV, (+++) indicates a strong WT1 PCR amplification product in the first step RT PCR, (++) indicates a WT1 amplification product that is detectable by first step WT1 RT PCR, (+) indicates a product that is detectable only in the second step of WT1 RT PCR, and (−) indicates WT1 PCR negative.

TABLE XLIX Detection of WT1 mRNA in Mouse Tumor Cell Lines Cell Line WT1 mRNA K562 (human leukemia; ATCC): Positive control; (Lozzio +++ and Lozzio, Blood 45:321–334, 1975) TRAMPC (SV40 transformed prostate, B6); Foster et al., +++ Cancer Res. 57:3325–3330, 1997 BLK-SV40 HD2 (SV40-transf. fibroblast, B6; ATCC); ++ Nature 276:510–511, 1978 CTLL (T-cell, B6; ATCC); Gillis, Nature 268:154–156, + 1977) FM (FBL-3 subline, leukemia, B6); Glynn and Fefer, + Cancer Res. 28:434–439, 1968 BALB 3T3 (ATCC); Aaroston and Todaro, J. Cell. + Physiol. 72:141–148, 1968 S49.1 (Lymphoma, T-cell like, B/C; ATCC); Horibata and + Harris, Exp. Cell. Res. 60:61, 1970 BNL CL.2 (embryonic liver, B/C; ATCC); Nature + 276:510–511, 1978 MethA (sarcoma, B/C); Old et al., Ann. NY Acad. Sci. − 101:80–106, 1962 P3.6.2.8.1 (myeloma, B/C; ATCC); Proc. Nati. Acad. − Sci. USA 66:344, 1970 P2N (leukemia, DBA/2; ATCC); Melling et al., J. − Immunol. 117:1267–1274, 1976 BCL1 (lymphoma, B/C; ATCC); Slavin and Strober, Nature − 272:624–626, 1977 LSTRA (lymphoma, B/C); Glynn et al., Cancer Res. − 28:434–439, 1968 E10/EL-4 (lymphoma, B6); Glynn et al., Cancer Res. − 28:434–439, 1968

Example 7 Expression in E. coli of WT-1 Trx Fusion Construct

The truncated open reading frame of WT-1 (WT-1B) was PCR amplified with the following primers:

Forward Primer starting at amino acid 2 P–37 (SEQ ID NO.347) 5′ggctccgacgtgcgggacctg 3′ Tm 64° C. Reverse Primer creating EcoRI site after stop co- don P–23 (SEQ ID NO.348) 5′gaattctcaaagcgccagctggagtttggt 3′ Tm 63° C.

The PCR was performed under the following conditions:

10 μl 10X Pfu buffer 1 μl 10 mM dNTPs 2 μl 10 μM each oligo 83 μL sterile water 1.5 μl Pfu DNA polymerase (Stratagene, La Jolla, CA) 50 ng DNA (pPDM FL WT-1) 96° C. 2 minutes 96° C. 20 seconds 63° C. 72° C. 15 seconds 3 minutes × 40 cycles 72° C. 4 minutes

The PCR product was digested with EcoRI restriction enzyme, gel purified and then cloned into pTrx 2H vector (a modified pET28 vector with a Trx fusion on the N-terminal and two His tags surrounding the Trx fusion. After the Trx fusion there exists protease cleavage sites for thrombin and enterokinase). The pTrx2H construct was digested with StuI and EcoRI restriction enzymes. The correct constructs were confirmed by DNA sequence analysis and then transformed into BL21 (DE3) pLys S and BL21 (DE3) CodonPlus expression host cells.

Example 8 Expression in E. coli of WT-1 a His Tag Fusion Constructs

The N-terminal open reading frame of WT-1 (WT-1A) was PCR amplified with the following primers:

Forward Primer starting at amino acid 2 P-37 (SEQ ID NO.349) 5′ggctccgacgtgcgggacctg 3′ Tm 64° C. Reverse Primer creating EcoRI site after an arti- ficial stop codon put after amino acid 249. PDM-335 (SEQ ID NO.345) 5′gaattctcaaagcgccagctggagtttggt 3′ Tm 64° C.

The PCR was performed under the following conditions:

10 μl 10X Pfu buffer 1 μl 10 mM dNTPs 2 μl 10 μM each oligo 83 μL sterile water 1.5 μl Pfu DNA polymerase (Stratagene, La Jolla, CA) 50 ng DNA (pPDM FL WT-1) 96° C. 2 minutes 96° C. 20 seconds 63° C. 72° C. 15 seconds 1 minute 20 seconds × 40 cycles 72° C. 4 minutes

The PCR product was digested with EcoRI restriction enzyme, gel purified and then cloned into pPDM, a modified pET28 vector with a His tag in frame, which had been digested with Eco72I and EcoRI restriction enzymes. The PCR product was also transformed into pTrx 2H vector. The pTrx2H construct was digested with StuI and EcoRI restriction enzymes. The correct constructs were confirmed by DNA sequence analysis and then transformed into BL21 (DE3) pLys S and BL21 (DE3) CodonPlus expression host cells.

Example 9 Expression in E. coli of WT-1 B His Tag Fusion Constructs

The truncated open reading frame of WT-1 (WT-1A) was PCR amplified with the following primers:

Forward Primer starting at amino acid 250 PDM-346 (SEQ ID NO.350) 5′cacagcacagggtacgagagc 3′ Tm 58° C. Reverse Primer creating EcoRI site after stop co- don P-23 (SEQ ID NO.347) 5′gaattctcaaagcgccagctggagtttggt 3′ Tm 63° C.

The PCR was performed under the following conditions:

10 μl 10X Pfu buffer 1 μl 10 mM dNTPs 2 μl 10 μM each oligo 83 μL sterile water 1.5 μl Pfu DNA polymerase (Stratagene, La Jolla, CA) 50 ng DNA (pPDM FL WT-1) 96° C. 2 minutes 96° C. 20 seconds 63° C. 72° C. 15 seconds 1 minute 30 seconds × 40 cycles 72° C. 4 minutes

The PCR product was digested with EcoRI restriction enzyme, gel purified and then cloned into pPDM, a modified pET28 vector with a His tag in frame, which had been digested with Eco72I and EcoRI restriction enzymes. The PCR product was also transformed into pTrx 2H vector. The pTrx 2H construct was digested with StuI and EcoRI restriction enzymes. The correct constructs were confirmed by DNA sequence analysis and then transformed into BL21 (DE3) pLys S and BL21 (DE3) CodonPlus expression host cells.

For Examples 7–9, the following SEQ ID NOs. are disclosed:

-   SEQ ID NO. 327 is the determined cDNA sequence for Trx_WT-1_B -   SEQ ID NO. 328 is the determined cDNA sequence for Trx_WT-1_A -   SEQ ID NO. 329 is the determined cDNA sequence for Trx_WT-1 -   SEQ ID NO. 330 is the determined cDNA sequence for WT-1_A -   SEQ ID NO. 331 is the determined cDNA sequence for WT-1_B -   SEQ ID NO. 332 is the predicted amino acid sequence encoded by SEQ     ID No. 327 -   SEQ ID NO. 333 is the predicted amino acid sequence encoded by SEQ     ID No. 328 -   SEQ ID NO. 334 is the predicted amino acid sequence encoded by SEQ     ID No. 329 -   SEQ ID NO. 335 is the predicted amino acid sequence encoded by SEQ     ID No. 330 -   SEQ ID NO. 336 is the predicted amino acid sequence encoded by SEQ     ID No. 331

Example 10 Truncated Forms of WT-1 Expressed in E. coli

Three reading frames of WT-1 were amplified by PCR using the following primers:

For WT-1 Tr2:

For WT-1 Tr2: PDM-441 (SEQ ID NO. 353) 5′ cacgaagaacagtgcctgagcgcattcac 3′ Tm 63° C. PDM-442 (SEQ ID NO. 354) 5′ ccggcgaattcatcagtataaattgtcactgc 3′ TM 62° C. For WT-1 Tr3: PDM-443 (SEQ ID NO. 355) 5′ caggctttgctgctgaggacgccc 3′ Tm 64° C. PDM-444 (SEQ ID NO. 356) 5′ cacggagaattcatcactggtatggtttctcacc Tm 64° C. For WT-1 Tr4: PDM-445 (SEQ ID NO. 357) 5′ cacagcaggaagcacactggtgagaaac 3′ Tm 63° C. PDM-446 (SEQ ID NO. 358) 5′ ggatatctgcagaattctcaaagcgccagc 3′ TM 63° C.

The PCR was performed under the following conditions:

10 μl 10X Pfu buffer 1 μl 10 mM dNTPs 2 μl 10 μM each oligo 83 μL sterile water 1.5 μl Pfu DNA polymerase (Stratagene, La Jolla, CA) 50 ng DNA (pPDM FL WT-1) 96° C. 2 minutes 96° C. 20 seconds 63° C. 72° C. 15 seconds 20 seconds × 40 cycles 72° C. 4 minutes

The PCR products were digested with EcoRI and cloned into pPDM His (a modified pET28 vector with a His tag in frame on the 5′ end) which has been digested with Eco72I and EcoRI. The constructs were confirmed to be correct through sequence analysis and transformed into BL21 pLys S and BL21 CodonPlus cells or BLR pLys S and BLR CodonPlus cells.

Example 11 WT-1 C (Amino Acids 76–437) and WT-1 D (Amino Acids 91–437) Expression in E. coli

The WT-1 C reading frame was amplified by PCR using the following primers:

PDM-504 (SEQ ID NO. 359) 5′ cactccttcatcaaacaggaac 3′ Tm 61° C. PDM-446 (SEQ ID NO. 360) 5′ ggatatctgcagaattctcaaagcgccagc 3′ Tm 63° C.

The PCR was performed under the following conditions:

10 μl 10X Pfu buffer 1 μl 10 mM dNTPs 2 μl 10 μM each oligo 83 μL sterile water 1.5 μl Pfu DNA polymerase (Stratagene, La Jolla, CA) 50 ng DNA (pPDM FL WT-1) 96° C. 2 minutes 96° C. 20 seconds 63° C. 72° C. 15 seconds 2 minutes × 40 cycles 72° C. 4 minutes

The PCR product was digested with EcoRI and cloned into pPDM His which had been digested with Eco72I and EcoRI. The sequence was confirmed through sequence analysis and then transformed into BLR pLys S and BLR which is co-transformed with CodonPlus RP.

Example 12 Synthetic Production of WT-1 TR-1 by Annealing Overlapping Oligos

This example was performed to determine the effect of changing proline codon usage on expression.

The following pairs of oligos were annealed:

1. PDM-505 (SEQ ID NO. 361) 5′ ggttccgacgtgcgggacctgaacgcactgctg 3′ PDM-506 (SEQ ID NO. 362) 5′ ctgccggcagcagtgcgttcaggtcccgcacgtcggaacc 3′ 2. PDM-507 (SEQ ID NO. 363) 5′ ccggcagttccatccctgggtggcggtggaggctg 3′ PDM-508 (SEQ ID NO. 364) 5′ cggcagtgcgcagcctccaccgccacccagggatggaa 3′ 3. PDM-509 (SEQ ID NO. 365) 5′ cgcactgccggttagcggtgcagcacagtgggctc 3′ PDM-510 (SEQ ID NO. 366) 5′ cagaactggagcccactgtgctgcaccgctaac 3′ 4. PDM-511 (SEQ ID NO. 367) 5′ cagttctggacttcgcaccgcctggtgcatccgcatac 3′ PDM-512 (SEQ ID NO. 368) 5′ cagggaaccgtatgcggatgcaccaggcggtgcgaagtc 3′ 5. PDM-513 (SEQ ID NO. 369) 5′ ggttccctgggtggtccagcacctccgcccgcaacgcc 3′ PDM-514 (SEQ ID NO. 370) 5′ ggcggtgggggcgttgcgggcggaggtgctggaccacc 3′ 6. PDM-515 (SEQ ID NO. 371) 5′ cccaccgcctccaccgcccccgcactccttcatcaaacag 3′ PDM-516 (SEQ ID NO. 372) 5′ ctaggttcctgtttgatgaaggagtgcgggggcggtgga 3′ 7. PDM-517 (SEQ ID NO. 373) 5′ gaacctagctggggtggtgcagaaccgcacgaagaaca 3′ PDM-518 (SEQ ID NO. 374) 5′ ctcaggcactgttcttcgtgcggttctgcaccaccccag 3′ 8. PDM-519 (SEQ ID NO. 375) 5′ gtgcctgagcgcattctgagaattctgcagat 3′ PDM-520 (SEQ ID NO. 376) 5′ gtgtgatggatatctgcagaattctcagaatgcg 3′

Each oligo pair was separately combined then annealed. The pairs were then ligated together and one μl of ligation mix was used for PCR conditions below:

10 μl 10X Pfu buffer 1 μl 10 mM dNTPs 2 μl 10 μM each oligo 83 μL sterile water 1.5 μl Pfu DNA polymerase (Stratagene, La Jolla, CA) 96° C. 2 minutes 96° C. 20 seconds 63° C. 72° C. 15 seconds 30 seconds × 40 cycles 72° C. 4 minutes

The PCR product was digested with EcoRI and cloned into pPDM His which had been digested with Eco72I and EcoRI. The sequence was confirmed and then transformed into BLR pLys S and BLR which is co-transformed with CodonPlus RP.

For examples 10–12, the following SEQ ID NOs. are disclosed:

-   SEQ ID NO:337 is the determined cDNA sequence for WT-1_Tr1SEQ ID     NO:338 is the determined cDNA sequence for WT-1_Tr2 -   SEQ ID NO:339 is the determined cDNA sequence for WT-1_Tr3 -   SEQ ID NO:340 is the determined cDNA sequence for WT-1_Tr4 -   SEQ ID NO:341 is the determined cDNA sequence for WT-1_C -   SEQ ID NO:342 is the predicted amino acid sequence encoded by SEQ ID     NO:337 -   SEQ ID NO:343 is the predicted amino acid sequence encoded by SEQ ID     NO:338 -   SEQ ID NO:344 is the predicted amino acid sequence encoded by SEQ ID     NO:339 -   SEQ ID NO:345 is the predicted amino acid sequence encoded by SEQ ID     NO:340 -   SEQ ID NO:346 is the predicted amino acid sequence encoded by SEQ ID     NO:341

The WT-1 C sequence represents a polynucleotide having the coding regions of TR2, TR3 and TR4.

The WT-1 TR-1 synthetic sequence represents a polynucleotide in which alternative codons for proline were substituted for the native codons, producing a polynucleotide capable of expressing WT-1 TR-1 in E. coli.

Example 13 Evaluation of the Systemic Histopathological and Toxicological Effects of WT1 Immunization in Mice

The purpose of this example is to analyze the immunogenicity and potential systemic histopathological and toxicological effects of WT1 protein immunization in a multiple dose titration in mice.

The experimental design for immunization of mice with WT1 protein is outlined in Table L.

TABLE L Experimental Desian of WT1 Immunization in Mice Histology Corixa Dose Total No. Group Group Treatment Description Level (Females) 1 0   No treatment 0 4 2 1.1 MPL-SE (adjuvants alone), 6×, 1 week apart 10 ug 4 3 1.2 MPL-SE, 3×, 2 weeks apart 10 ug 4 4 2.1 Ral2-WT1 + MPL-SE, 6× 25 ug 4 5 2.2 Ral2-WT1 + MPL-SE, 3× 25 ug 4 6 3.1 Ral2-WT1 + MPL-SE, 6× 100 ug 4 7 3.2 Ral2-WT1 + MPL-SE, 3× 100 ug 4 8 4.1 Ral2-WT1 + MPL-SE, 6× 1000 ug 4 9 4.2 Ral2-WT1 + MPL-SE, 3× 1000 ug 4

Vaccination to WT1 protein using MPL-SE as adjuvant, in a multiple dose titration study (doses ranging from 25 μg, 100 μg to 1000 μg WT1 protein) in female C57/B6 mice elicited a strong WT1-specific antibody response (FIG. 19) and cellular T-cell responses (FIG. 20).

No systemic histopathological or toxicological effects of immunization with WT1 protein was observed. No histological evidence for toxicity was seen in the following tissues: adrenal gland, brain, cecum, colon, duodenum, eye, femur and marrow, gall bladder, heart, ileum, jejunum, kidney, larynx, lacrimal gland, liver, lung, lymph node, muscle, esophagus, ovary, pancreas, parathyroid, salivary gland, sternum and marrow, spleen, stomach, thymus, trachea, thyroid, urinary bladder and uterus.

Special emphasis was put on evaluation of potential hematopoietic toxicity. The myeloid/erythroid ratio in sternum and femur marrow was normal. All evaluable blood cell counts and blood chemistry (BUN, creatinine, bilirubin, albumin, globulin) were within the normal range (Table LI).

Given that existent immunity to WT1 is present in some patients with leukemia and that vaccination to WT1 protein can elicit WT1 specific Ab and cellular T-cell responses in mice without toxicity to normal tissues, these experiments validate WT1 as a tumor/leukemia vaccine.

TABLE LI WT1 Dose Titration Study Clinical Chemistry and Hematology Analysis K/uL M/uL g/dl % fL pg % Animal # WBC RBC Hg. HCT MCV MCH MCHC Normal 5.4–16.0 6.7–12.5 10.2–16.6 32–54 31–62 9.2–20.8 22.0–35.5 Group 1  1 (0) 5.6 8.41 12.8 43.5 53 15.2 29.4  2 (0) 5.5 9.12 13.4 47.5 53 14.7 28.2  3 (0) 7.5 9.22 13.5 48 54 14.7 28.1  4 (0) 3.9 9.27 13.6 46 52 14.7 29.6 Mean 5.6 9.0 13.3 46.3 53.0 14.8 28.8 STD 1.5 0.4 0.4 2.0 0.8 0.3 0.8 Group 2  5 (1.5) 6.6 9 13.1 46 54 14.5 28.5  6 (1.6) 5.2 8.58 12.6 44 53 14.7 28.6  7 (1.7) 7.8 9.21 13.6 46 53 14.7 29.6  8 (1.8) 6.3 NA NA 41 NA NA NA Mean 6.5 8.9 13.1 44.3 53.3 14.6 28.9 STD 1.1 0.3 0.5 2.4 0.6 0.1 0.6 Group 3  9 (2.5) 8.3 9.16 13.6 50.3 55 14.9 27.1 10 (2.6) 5 8.78 13 44.2 50 14.8 29.3 11 (2.7) 4 8.94 13.2 48.3 54 14.7 27.3 12 (2.8) 8.2 NA NA 41 NA NA NA Mean 6.4 9.0 13.3 46.0 53.0 14.8 27.9 STD 2.2 0.2 0.3 4.2 2.6 0.1 1.2 Group 4 13 (3.5) 6.1 8.82 13.1 46 54 14.9 28.5 14 (3.6) 6.1 8.64 12.9 46 54 15 28 15 (3.7) 9.3 8.93 13.2 48 55 14.8 27.5 16 (3.8) 4.8 8.19 12.6 44 55 15.3 28.6 Mean 6.6 8.6 13.0 46.0 54.5 15.0 28.2 STD 1.9 0.3 0.3 1.6 0.6 0.2 0.5 Group 5 17 (4.5) 3.1 8.48 12.6 46 54 14.9 27.5 18 (4.6) 5.7 9.12 13.7 48 54 15 28.5 19 (4.7) 5.3 8.58 13 44.5 55 15.2 29.2 20 (4.8) 5.3 NA NA 40 NA NA NA Mean 4.9 8.7 13.1 44.6 54.3 15.0 28.4 STD 1.2 0.3 0.6 3.4 0.6 0.2 0.9 Group 6 21 (1.1) 3.5 9.36 13.5 37.6 40 14.4 35.9 22 (1.2) 6.9 8.93 13.6 37.3 42 15.3 36.6 23 (1.3) 3.6 8.3 12.5 35.3 43 15.1 35.5 24 (1.4) NA NA NA NA NA NA NA Mean 4.7 8.9 13.2 36.7 41.7 14.9 36.0 STD 1.9 0.5 0.6 1.3 1.5 0.5 0.6 Group 7 25 (2.1) 4 NA NA 40 NA NA NA 26 (2.2) 7.4 9.12 13.2 38.5 42 14.5 34.3 27 (2.3) 4.5 8.19 12.1 34.5 42 14.8 35.1 28 (2.4) 5.8 8.25 12.3 34.1 41 14.9 36.1 Mean 5.4 8.5 12.5 36.8 41.7 14.7 35.2 STD 1.5 0.5 0.6 2.9 0.6 0.2 0.9 Group 8 29 (3.1) 5.1 8.53 12.6 34.9 41 14.7 36 30 (3.2) 7.6 8.42 13 36.1 43 15.4 35.9 31 (3.3) 3.4 8.45 12.6 34.9 41 14.9 36.1 32 (3.4) 6.1 8.11 12.3 34.8 43 15.2 35.5 Mean 5.6 8.4 12.6 35.2 42.0 15.1 35.9 STD 1.8 0.2 0.3 0.6 1.2 0.3 0.3 Group 9 33 (4.1) NA NA NA NA NA NA NA 34 (4.2) 4.5 8.63 12.8 36.2 42 14.8 35.2 35 (4.3) 3.9 8.85 13 36.6 41 14.7 35.6 36 (4.4) 4.7 8.14 12.3 33.8 42 15.1 36.3 Mean 4.4 8.5 12.7 35.5 41.7 14.9 35.7 STD 0.4 0.4 0.4 1.5 0.6 0.2 0.6 yes/no K/uL Abs. Abs. Abs. Abs. Abs. Abs. Animal # Plt. clump Platelets Baso Eos Bands Polys Lymph Mono Normal no 150–1500 0.0–0.15 0.0–0.51 0.0–0.32 8.0–42.9 8.0–18.0 0.0–1.5 Group 1 K/uL K/uL K/uL K/uL K/uL K/uL  1 (0) yes 726 0 56 0 336 5208 0  2 (0) no 860 0 0 0 55 5445 0  3 (0) no 875 0 375 0 525 6525 75  4 (0) yes 902 0 0 0 156 3744 0 Mean 840.8 0.0 107.8 0.0 268.0 5230.5 18.8 STD 78.4 0.0 180.1 0.0 207.0 1144.8 37.5 Group 2  5 (1.5) no 1193 0 132 0 792 5214 462  6 (1.6) no 1166 0 52 0 624 4472 52  7 (1.7) no 1087 0 234 0 1170 6396 0  8 (1.8) yes NA 0 126 0 126 5922 126 Mean 1148.7 0.0 136.0 0.0 678.0 5501.0 160.0 STD 55.1 0.0 74.8 0.0 433.1 840.5 207.9 Group 3  9 (2.5) no 705 0 166 0 664 7387 83 10 (2.6) no 1140 0 150 0 500 4350 0 11 (2.7) no 952 0 120 0 680 3200 0 12 (2.8) yes NA 0 164 0 656 7216 164 Mean 932.3 0.0 150.0 0.0 625.0 5538.3 61.8 STD 218.2 0.0 21.2 0.0 83.9 2090.6 78.6 Group 4 13 (3.5) no 785 0 488 0 732 4636 244 14 (3.6) yes 973 0 0 0 488 5307 305 15 (3.7) yes 939 0 465 0 558 7812 465 16 (3.8) yes 1622 0 192 0 480 4080 48 Mean 1079.8 0.0 286.3 0.0 564.5 5458.8 265.5 STD 370.6 0.0 233.4 0.0 117.0 1647.1 172.4 Group 5 17 (4.5) no 892 0 31 0 620 2449 0 18 (4.6) yes 966 57 114 0 855 4674 0 19 (4.7) yes 883 0 53 0 742 4452 53 20 (4.8) yes NA 0 106 0 53 5141 0 Mean 913.7 14.3 76.0 0.0 567.5 4179.0 13.3 STD 45.5 28.5 40.4 0.0 356.2 1188.5 26.5 Group 6 21 (1.1) yes 784 0 35 0 385 2870 210 22 (1.2) yes 806 0 69 0 207 6486 138 23 (1.3) yes 790 0 180 0 396 2988 36 24 (1.4) NA NA NA NA NA NA NA NA Mean 793.3 0.0 94.7 0.0 329.3 4114.7 128.0 STD 11.4 0.0 75.8 0.0 106.1 2054.5 87.4 Group 7 25 (2.1) yes NA 0 80 0 200 3720 0 26 (2.2) yes 753 0 0 0 518 6734 148 27 (2.3) yes 725 0 90 0 225 4140 45 28 (2.4) yes 792 0 232 0 754 4814 0 Mean 756.7 0.0 100.5 0.0 424.3 4852.0 48.3 STD 33.7 0.0 96.5 0.0 263.0 1333.1 69.8 Group 8 29 (3.1) yes 784 0 153 0 561 4233 153 30 (3.2) yes 512 0 152 0 304 6992 152 31 (3.3) yes 701 0 0 0 238 3094 68 32 (3.4) yes 631 0 305 0 305 5368 122 Mean 657.0 0.0 152.5 0.0 352.0 4921.8 123.8 STD 115.1 0.0 124.5 0.0 142.8 1663.3 39.9 Group 9 33 (4.1) NA NA NA NA NA NA NA NA 34 (4.2) yes 724 0 125 0 540 3780 45 35 (4.3) yes 758 0 117 0 429 3315 39 36 (4.4) yes 808 0 47 0 329 4089 235 Mean 763.3 0.0 96.3 0.0 432.7 3728.0 106.3 STD 42.3 0.0 42.9 0.0 105.5 389.6 111.5 mg/dl Animal # mg/dl BUN mg/dl Creatinine g/dl T. protein g/dl Albumin g/dl Globulin T. Bilirubin Normal 13.9–28.3 0.3–1.0 4.0–8.6 2.5–4.8 1.5–3.8 0.10–0.90 Group 1  1 (0) NA NA NA NA NA NA  2 (0) 28 0.5 4.9 3.7 1.2 0.3  3 (0) 25 0.5 4.9 3.8 1.1 0.2  4 (0) 27 0.5 4.7 3.7 1 0.2 Mean 26.7 0.5 4.8 3.7 1.1 0.2 STD 1.5 0.0 0.1 0.1 0.1 0.1 Group 2  5 (1.5) 34 0.5 4.6 3.6 1 0.2  6 (1.6) 31 0.4 4.6 3.3 1.3 0.2  7 (1.7) 34 0.6 4.9 4 0.9 0.3  8 (1.8) NA NA NA NA NA NA Mean 33.0 0.5 4.7 3.6 1.1 0.2 STD 1.7 0.1 0.2 0.4 0.2 0.1 Group 3  9 (2.5) NA NA NA NA NA NA 10 (2.6) 33 0.5 4.6 3.6 1 0.3 11 (2.7) NA NA NA NA NA NA 12 (2.8) 31 0.5 4.8 3.7 1.1 0.2 Mean 32.0 0.5 4.7 3.7 1.1 0.3 STD 1.4 0.0 0.1 0.1 0.1 0.1 Group 4 13 (3.5) 32 0.7 4.6 3.4 1.2 0.2 14 (3.6) 34 0.4 4.8 3.8 1 0.2 15 (3.7) 30 0.4 4.7 3.4 1.3 0.2 16 (3.8) 24 0.3 5.1 3.8 1.3 0.2 Mean 30.0 0.5 4.8 3.6 1.2 0.2 STD 4.3 0.2 0.2 0.2 0.1 0.0 Group 5 17 (4.5) 22 0.4 4.6 3.3 1.3 0.2 18 (4.6) 31 0.5 4.9 3.7 1.2 0.2 19 (4.7) 23 0.6 4.8 3.6 1.2 0.2 20 (4.8) 28 0.5 4.5 3.4 1.1 0.2 Mean 26.0 0.5 4.7 3.5 1.2 0.2 STD 4.2 0.1 0.2 0.2 0.1 0.0 Group 6 21 (1.1) 28 0.3 5.1 3.4 1.7 0.2 22 (1.2) 36 0.3 5.1 3.8 1.3 0.2 23 (1.3) 32 0.4 4.9 3.5 1.4 0.1 24 (1.4) NA NA NA NA NA NA Mean 32.0 0.3 5.0 3.6 1.5 0.2 STD 4.0 0.1 0.1 0.2 0.2 0.1 Group 7 25 (2.1) 32 0.2 5 3.4 1.6 0.2 26 (2.2) 24 0.3 4.2 2.8 1.4 0.1 27 (2.3) 28 0.3 4.8 3.2 1.6 0.2 28 (2.4) 27 0.3 5 3.4 1.6 0.1 Mean 27.8 0.3 4.8 3.2 1.6 0.2 STD 3.3 0.0 0.4 0.3 0.1 0.1 Group 8 29 (3.1) 32 0.3 4.9 3.3 1.6 0.2 30 (3.2) NA NA NA NA NA NA 31 (3.3) 18 0.3 4.8 3.1 1.7 0.2 32 (3.4) 26 0.2 4.2 2.9 1.3 0 Mean 25.3 0.3 4.6 3.1 1.5 0.1 STD 7.0 0.1 0.4 0.2 0.2 0.1 Group 9 33 (4.1) 25 0.2 4.1 2.7 1.4 0.3 34 (4.2) NA NA NA NA NA NA 35 (4.3) 23 0.2 4.7 3.1 1.6 0.2 36 (4.4) 29 0.3 4.7 3.2 1.5 0.3 Mean 25.7 0.2 4.5 3.0 1.5 0.3 STD 3.1 0.1 0.3 0.3 0.1 0.1 Abbreviations: WBC: white blood cells; RBC: red blood cells; Hg.: hemoglobin; HCT: hematocrit; MCV: Mean corpuscular volume; MCH: mean corpuscular hemoglobin; MCHC: mean corpuscular hemoglobin concentration; Plt.: platelets; Abs.: Absolute; Baso: basophils; Eos: eosinophils; Abs. Bands: immature neutrophils; Polys: polymorphonuclear cells; Lymph: lymphocytes; Mono: monocytes; BUN: blood urea nitrogen

Example 14 Elicitation of Human WT1-specific T-cell Responses by Whole Gene in vitro Priming

This example demonstrates that WT1 specific T-cell responses can be generated from the blood of normal individuals.

Dendritic cells (DC) were differentiated from monocyte cultures derived from PBMC of normal donors by growth for 4–10 days in RPMI medium containing 10% human serum, 50 ng/ml GMCSF and 30 ng/ml IL-4. Following culture, DC were infected 16 hours with recombinant WT1-expressing vaccinia virus at an M.O.I. of 5, or for 3 days with recombinant WT1-expressing adenovirus at an M.O.I. of 10 (FIGS. 21 and 22). Vaccinia virus was inactivated by U.V. irradiation. CD8+ T-cells were isolated by positive selection using magnetic beads, and priming cultures were initiated in 96-well plates. Cultures were restimulated every 7–10 days using autologous dendritic cells adeno or vaccinia infected to express WT1. Following 3–6 stimulation cycles, CD8+ lines could be identified that specifically produced interferon-gamma when stimulated with autologous-WT1-expressing dendritic cells or fibroblasts. The WT1-specific activity of these lines could be maintained following additional stimulation cycles. These lines were demonstrated to specifically recognize adeno or vaccinia WT1 infected autologous dendritic cells but not adeno or vaccinia EGFP-infected autologous dendritic cells by Elispot assays (FIG. 23).

Example 15 Formulation of RA12-WT1 For Injection: Use of Excipients to Stabilize Lyophilized Product

This example describes the formulation that allows the complete solubilization of lyophilized Ra12-WT1.

The following formulation allowed for the recombinant protein Ra12-WT1 to be dissolved into an aqueous medium after being lyophylized to dryness:

Recombinant Ra12-WT1 concentration: 0.5–1.0 mg/ml; Buffer: 10–20 mM Ethanolamine, pH 10.0; 1.0–5.0 mM Cysteine; 0.05% Tween-80 (Polysorbate-80); Sugar: 10% Trehalose (T5251, Sigma, Mo.) 10% Maltose (M9171, Sigma, Mo.) 10% Sucrose (S7903, Sigma, MO) 10% Fructose (F2543, Sigma, MO) 10% Glucose (G7528, Sigma, MO).

The lyophilized protein with the sugar excipient was found to dissolve significantly more than without the sugar excipient. Analysis by coomassie stained SDS-PAGE showed no signs of remaining solids in the dissolved material.

From the foregoing it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims. 

1. A polypeptide consisting of the amino acid sequence of SEQ ID NO:335.
 2. A composition comprising the polypeptide of claim 1 in combination with a pharmaceutically acceptable carrier or excipient.
 3. A composition comprising the polypeptide of claim 1 in combination with a non-specific immune response enhancer.
 4. The composition according to claim 3 wherein the non-specific immune response enhancer preferentially enhances a T cell response in a patient.
 5. The composition according to claim 3, wherein the immune response enhancer comprises a microsphere. 